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Optimisation of expression of recombinant

puroindolines and analysis of

protein-protein interactions in planta

Azadeh Niknejad

This thesis is presented for the degree of

Doctor of Philosophy

June 2014

Department of Chemistry and Biotechnology

Faculty of Science, Engineering and Technology

Swinburne University of Technology

Melbourne, Australia

i

Abstract

The two puroindoline proteins of wheat (Triticum aestivum L.), puroindoline-a (PINA)

and puroindoline-b (PINB) are largely responsible for grain texture, a property

important in food technology and the wheat trade. Furthermore, it has been suggested

that PINs may have an in vivo role in seed pathogen defence and antimicrobial

properties, which make them very attractive as antimicrobial agents for novel medical,

pharmaceutical and food-industry applications. Soft grain texture in wheat grains

depends on the presence of both PIN proteins in their wild type and functional form.

Variations in either or both genes, or lack of expression of either gene, result in a hard

grain texture. Currently, the biochemical basis of the role of PIN proteins in endosperm

texture as well as in antimicrobial functions remains unclear. Plant expression systems

are being developed to produce recombinant PINA and PINB proteins, with the aim to

assess their structure and function. For therapeutic use, production and purification of

high quality PIN proteins is important. One aspect of this study focussed on the rapid

transient expression of PINs for better yield using the deconstructed tobacco mosaic

virus-based ‘magnICON®’ plant expression system. This system was used to test the

subcellular localisation of PINs in different compartments of Nicotiana benthamiana

cells (cytosol, chloroplast, apoplast and endoplasmic reticulum). The results indicated a

profound impact of the cellular compartment on the yield and stability of the PIN

proteins. The presence of recombinant PINA and PINB was confirmed using western

blot and ELISA analyses. Maximum yields of His-tagged recombinant PINA and PINB

occurred when proteins were targeted to the chloroplast. Each PIN protein was shown

to occur in both monomeric and oligomeric forms, which addresses some of the

observations in the literature related to their functionality in determining wheat grain

texture. Affinity purification systems using His-tag and hydrophobic interactions were

used to purify the recombinant PINs. The purified proteins exhibited antibacterial and

antifungal activities, suggesting they were correctly folded. There is evidence that PINs

may interact co-operatively or interdependently to confer the soft grain texture and

influence in pathogen protection. Therefore, as a second major focus of this work,

interactions between PINs were investigated using the Bimolecular Fluorescence

Complementation (BiFC) system in a plant system. Results based on the fluorescence

intensity obtained and confirmed in vivo interactions between PINA and PINB and also

between PINB and PINB in planta. In order to investigate the regions involved in the

ii

protein-protein interactions of PINs, clones constructed previously containing deletion

of the tryptophan-rich domain (TRD) in PINA and the hydrophobic domain (HD) in

both PINs were used for cloning into BiFC vectors. The result indicated that no

interaction occurred between the mutant PIN proteins. The finding shows that both the

TRD and HD regions may have roles in the interactions of PIN proteins, but may

contribute in different ways to their folding and stability.

The work has led to successful expression, purification and characterisation

methodology for obtaining PINs of appropriate quality essential for potential infection

control in diverse areas. Additionally, the work also confirmed their in planta

interactions. To our knowledge, this is the first report of functional PINs produced in N.

benthamiana based on transient expression, and it provides a starting point for

investigating a potential role for PINs on grain texture and antimicrobial abilities.

iii

Acknowledgments

All praises to Almighty God for the strength and His blessing in completing this work.

I would like to thank my principal supervisor, Professor Mrinal Bhave for giving me the

great opportunity of performing this research and for her support, guidance and patience

throughout this project. Thanks particularly for all the time that she has put on the

preparation of this thesis. I would also like to express my deepest appreciation and

gratitude to Dr Diane Webster for acting as a supervisor and being helpful and

supportive from the first day I stepped into the Plant Biotechnology laboratory in

Monash University for three years. Thanks for encouragement and generosity of time in

all meetings during this study.

I am also grateful to Swinburne University of Technology for awarding me the SUPRA

scholarship.

Thanks to Professor Michael Gilding and Dr Elena Verezub for advice and help and Ms

Angela McKellar in Swinburne University. I would also like to thank Professor David

Smyth and Mr Tezz Quon in Monash University for providing plasmid pNBV and

pMLBART. Thanks to Associate Professor Sureshkumar Balasubramanian and his

group for always welcoming me in SKB lab in Monash University.

Thank you to all my lab mates and friends in Monash University; Rasika, Kartika,

Claire, Robert, Indramohan, David, Vincent and Victor.

Thank you to past and present PhD students in Swinburne University; Dr Peter Gollan

and Dr Rebecca Alfred for always sharing their knowledge. Many thanks to Dr

Abirami Ramalingam, Dr Runyararo Hove, Dr Shanthi Joseph and Dr Kaylass Poorun

for all their help, encouragement and friendship. Thank you to my other friends; Bita,

Narges, Farnaz, Saifone, Atul, Yen, Guri, Nadin, Rasika, Jafar, June, Elisa, Dhivya,

Snehal, Rashida and Chris.

iv

Dedicated to

The memory of my dear father; Rasoul

and

My beloved mother; Zahra

For her loving support and always believing in me

v

Declaration

I, Azadeh Niknejad, declare that the PhD thesis entitled ‘Optimisation of expression of

recombinant puroindolines and analysis of protein-protein interactions in planta’ is no

more than 100,000 words in length, exclusive of tables, figures, appendices, references

and footnotes. This thesis contains no material that has been submitted previously, in

whole or in part, for the award of any other academic degree or diploma, and has not

been previously published by another person. Except where otherwise indicated, this

thesis is my own work.

Azadeh Niknejad

June 2014

vi

Abbreviations

BDT Big Dye Terminator

BiFC Bimolecular Fluorescence Complementation

bp Base pair

BSA Bovine Serum Albumin

C-terminal Carboxyl terminal (of a protein)

Cys (or C) Cysteine

DNA Deoxyribonucleic acid

dNTP Deoxyribonucleotide (A, T, G or C)

dpi Days post infiltration

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme linked immunosorbent assay

ER Endoplasmic Reticulum

g Centrifuge force

gDNA Genomic DNA

GFP Green Fluorescent Protein

GSP-1 Grain Softness Protein-1 (a component of friabilin)

His (or H) Histidine

IPTG Isopropyl β-D-1-thiogalactopyranoside

Kb Kilobase pairs; 1kb= 1000 bp

kDa KiloDalton

LB Luria Bertani broth

MALDI-TOF Matrix-assisted laser desorption ionisation-time of flight

mg Milligram

mL Millilitre

MW Molecular Weight

N-terminal Amino terminal (of a protein)

OD Optical Density

ORF Open Reading Frame

PCR Polymerase Chain Reaction

Pin Puroindoline genes

Pina Puroindoline-a gene

Pinb Puroindoline-b gene

vii

PIN(s) Puroindoline protein(s)

PINA Puroindoline-a protein (wild type)

PINB Puroindoline-b protein (wild type)

rPINA Recombinant PINA

rPINB Recombinant PINB

PPI Protein-Protein Interaction

RBCs Red Blood Cells

rpm Revolutions per minute

SDM Site-directed mutagenesis

SDS-PAGE Sodium dodecyl sulphate-poly acrylamide gel electrophoresis

TAE Tris acetate ethlenediaminetetraacetic acid buffer

TBSV Tomato Bushy Stunt Virus

Trp (or W) Tryptophan

TRD Tryptophan-rich domain

TSP Total Soluble Protein

UV Ultra Violet

X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside

YFP Yellow Fluorescent Protein

YC C-terminal fragment of YFP

YN N-terminal fragment of YFP

°C Degree Celsius

List of databases/softwares

ApE A plasmid Editor

BioEdit Biological sequence alignment editor

BLAST Basic Local Alignment Search Tool

COTH Threading-recombination approach

ExPASY Expert Protein Analysis System

I-TASSER Iterative Threading Assembly Refinement

NCBI National Center for Biotechnology Information

PyMOL Python-enhanced molecular graphics tool

WoLF PSORT PSORT II program for protein subcellular location prediction

viii

Table of contents

Chapter 1 General introduction and literature review 1

1 General introduction 2

1.1 Introduction to wheat 2

1.1.1 Evolution of wheat species 3

1.1.2 Structure of the wheat grain 5

1.2 Grain texture in wheat 6

1.2.1 Friabilin 7

1.3 Puroindoline genes and grain texture 8

1.3.1 Mutations, duplications and deletions in Pin genes 8

1.3.2 Point mutations, duplications and deletions in Pina-D1 gene 9

1.3.3 Point mutations, duplications and deletions in Pinb-D1 gene 9

1.3.4 Pinb-2 genes 10

1.3.5 Pin-like genes in related species 12

1.4 Biochemical properties and structure of PIN proteins 13

1.4.1 Heterologous expression of recombinant PIN proteins 18

1.4.2 Evidence of transgenic Pin genes altering grain texture 20

1.5 Evidence of in vitro antimicrobial activity of PINs 22

1.5.1 Evidence of in vivo antimicrobial activity of PINs 23

1.5.2 Evidence of antimicrobial activity of PIN-based peptides 25

1.5.3 Possible mode of action for PINs as antimicrobial proteins and

peptides

25

1.5.4 Possible interactions between PINs 29

1.6 Plant expression system 30

1.6.1 Host plants 31

1.6.1.1 Nicotiana tabacum (Tobacco) 31

1.6.1.2 Nicotiana benthamiana 32

1.6.2 Transformation method and expression systems 32

1.6.2.1 Transient expression using viral vectors 33

1.6.3 Subcellular location in plant system for protein accumulation 34

1.6.3.1 Cytosol 35

1.6.3.2 Endoplasmic Reticulum (ER) 35

ix

1.6.3.3 Apoplast 35

1.6.3.4 Vacuole 35

1.6.3.5 Chloroplast 36

1.6.4 Plant systems for heterologous production of antimicrobial

peptide

36

1.7 Summary of the literature and aims of the research 39

1.7.1 Summary of the above literature 39

1.7.2 The aims of the project 40

Chapter 2 Materials and methods 41

Equipment and materials

2.1 Equipment 42

2.2 Commercial kits 43

2.3 Primary and secondary antibodies 44

2.4 Commonly used buffers, other solutions and media 44

2.4.1 Protein purification buffers 46

2.5 Microbial strains 46

2.5.1 Strains used as hosts for gene cloning 46

2.5.2 Strains used for testing of antimicrobial activity of

recombinant PINs

47

2.6 Plant material 47

2.6.1 Propagation of wheat seedlings 47

2.6.2 Propagation of Nicotiana benthamiana seedlings 47

2.7 General molecular methods 48

2.7.1 Genomic DNA extraction 48

2.7.2 Plasmid DNA purification 49

2.7.3 Spectrophotometric quantification of DNA 49

2.7.4 Agarose gel electrophoresis 49

2.8 The polymerase chain reactions (PCR) for gene amplifications 50

2.8.1 Design and synthesis of primers 50

2.8.2 Design of primers for amplification of full-length Pin genes 50

2.8.3 Design of primers for directional cloning into magnICON®

and BiFC vectors

51

2.8.4 Typical PCR conditions 51

x

2.8.5 PCR for directional cloning of Pin genes into plant expression

vectors

51

2.8.6 Purification of PCR products 52

2.9 Cloning and DNA sequencing 52

2.9.1 Restriction enzyme digestions of PCR products and vectors 52

2.9.2 Ligation reactions 53

2.9.3 Preparation of chemically competent cells of E. coli Mach1 53

2.9.4 Transformation of E. coli Mach1 competent cells by heat

shock

54

2.9.5 Screening of E. coli transformants containing recombinant

plasmids by colony PCR

54

2.9.6 Preparation of electro-competent A. tumefaciens cells 54

2.9.7 Electroporation of electro-competent A. tumefaciens cells 55

2.9.8 Screening of A. tumefaciens transformants containing

recombinant plasmids by colony PCR

55

2.9.9 DNA sequencing 56

2.10 Bioinformatics methods 56

2.10.1 Sequence alignments 56

2.10.2 Plasmid vector map 56

2.10.3 Predictions of the isoelectric point (pI) and molecular weight

(Mw) of PIN proteins

57

2.10.4 Prediction of protein subcellular localisation 57

2.10.5 Modeling and structure prediction of proteins 57

2.10.6 Protein-Protein complexes structure prediction 58

2.11 Biochemical methods for protein analysis 58

2.11.1 Protein concentration measurements 58

2.11.2 SDS-PAGE 59

2.11.3 Western blotting analysis for PIN proteins detection 59

2.11.4 Enzyme-linked immunosorbent assay (ELISA) for

approximate quantitation of recombinant PIN proteins

60

2.12 Testing of antimicrobial properties of the in planta expressed

PINs and a PIN-based synthetic peptide

61

2.12.1 Design of synthetic peptide based on PINA 61

xi

2.12.2 Antibacterial activity assay 61

2.12.3 Antifungal activity assay 62

2.12.4 Stability testing of recombinant PIN proteins 63

2.12.5 Haemolytic activity assay 63

Methods specific to Chapter 3

2.13 Expression of PIN proteins in N. benthamiana using

magnICON® viral vector and characterisation of their

biochemical and antimicrobial properties

64

2.13.1 Principles of the magnICON® viral vector systems for in

planta expression

64

2.13.2 Design of primers for directional cloning into the 3’ module

pICH11599

67

2.13.3 Amplifications of Pin genes, restriction enzyme digestion,

cloning into the 3’ module pICH11599 and sequencing of

clones

68

2.13.4 Electroporation of recombinant pICH11599 and other

modules into A. tumefaciens

70

2.13.5 Agroinfiltration of N. benthamiana using the magnICON®

viral vectors

71

2.13.6 GFP expression 72

Protein methods

2.13.7 Extraction of total soluble protein (TSP) from N. benthamiana

leaf

72

2.13.8 Protein concentration measurements, SDS-PAGE, western

blotting for detection and ELISA for approximate quantitation

of PINs

73

Methods for further PIN protein purification

2.13.9 His-tag purification under native conditions 73

2.13.10 His-tag purification under denaturing conditions 74

2.13.11 Hydrophobic interaction purification 74

2.13.12 Dialysis 75

2.13.13 Concentrate of dilute protein samples 75

2.13.14 Sodium phytate precipitation 75

xii

2.13.15 Testing of antimicrobial and haemolytic properties of the

expressed PINs

76

Methods specific to Chapter 4:

2.14 Protein-protein interactions of PIN proteins using BiFC

system

76

2.14.1 Principle of the Bimolecular Fluorescence Complementation

(BiFC) system

76

2.14.2 New BiFC Vector (pNBV) 77

2.14.3 Design of primers for directional cloning into BiFC vectors

(pNBVs)

78

2.14.4 Restriction enzyme digestion of PCR products 79

2.14.5 Cloning of PIN-YFP fragments into pMLBART vector 80

2.14.6 Electroporation into A. tumefaciens GV3101 81

2.14.7 Use of TBSV-P19 vector 81

2.14.8 Agroinfiltration of N. benthamiana using BiFC vectors 82

2.14.9 Fluorescence microscope and imaging 82

2.14.10 Protein methods for detection of BiFC 82

2.14.11 Native gel electrophoresis and detection of YFP fluorescence 83

2.15 Identification of any protein-protein interaction regions of

PINs using BiFC

85

2.15.1 Gifts of gene construct containing deletions of the tryptophan-

rich domain (TRD) of PINA and the hydrophobic domain

(HD) of PINA and PINB

85

2.15.2 Cloning of PINA∆TRD and PINA∆HD and PINB∆HD in

pNBV vectors

86

2.15.3 Cloning of PINA∆TRD and PINA∆HD and PINB∆HD into

the 3’ module pICH11599

87

Chapter 3 Results 88

3.1 Introduction 89

3.2 Expression of PINs using magnICON® viral vectors 91

3.2.1 Cloning PINA and PINB into magnICON® viral vector

system

91

3.2.2 Transient expression of PIN proteins in the viral vector system 94

xiii

3.2.3 Expression of Green Fluorescent Protein (GFP) by the viral

vector system

95

3.2.4 Expression of the recombinant PINA protein by the viral

vector system

97

3.2.5 Expression of the recombinant PINB protein by the viral

vector system

100

3.2.6 Optimisations of expression of His-tagged recombinant PINA

and PINB

102

3.3 Results of purification of PINA and PINB 105

3.3.1 His-tag purification under native conditions 105

3.3.2 His-tag purification under denaturing conditions 107

3.3.3 Hydrophobic interaction purification 108

3.3.4 Western blot analysis using anti His-tag for PIN proteins 111

3.4 Results of antimicrobial activity tests 112

3.4.1 Antibacterial activity 112

3.4.2 Antifungal activity 114

3.4.3 Protein stability at different temperature 116

3.4.4 Haemolytic activity 117

3.5 Discussion 118

3.5.1 Expression 118

3.5.2 Purification 120

3.5.3 Characterisation of bioactivity 122

Chapter 4 Results 125

4.1 Introduction 126

4.2A Interaction studies for wild type PINA and PINB 127

4.2.1 Cloning of wild type PINA and PINB into pNBV vectors of

the BiFC system

127

4.2.2 BiFC system for transient expression in N. bethamiana leaves 132

4.2.3 Post-transcriptional gene silencing (PTGS) in transient system 132

4.2.4 Fluorescence detection for wild type PIN protein-protein

interactions

133

4.2.5 Western blot analysis of wild type PIN protein-protein

interactions

135

xiv

4.2.6 Gel detection of YFP fluorescence for wild type PIN protein-

protein interactions

136

4.2.B Interaction studies of mutagenesed PINA and PINB 137

4.2.7 Cloning of the previously made constructs PINA∆HD and

PINB∆HD into pNBV vectors

137

4.2.8 Detection of mutant PINA∆HD and PINB∆HD protein-

protein interactions by fluorescence and western blot

141

4.2.9 Cloning of PINA∆TRD into pNBV vectors 142

4.2.10 Fluorescence detection for mutant PINA∆TRD protein-protein

interactions

144

4.2.11 Expression of mutant PIN proteins in magnICON® system 145

4.2.12 Bioinformatics analysis of PIN sequences for potential

interaction domain(s)

149

4.3 Discussion 155

Chapter 5 General discussion and future directions 159

5.1 General discussion 160

5.2 Future directions 163

References 165

Appendices 200

xv

List of Figures

1.1 Schematic representation of the evolutionary history of wheat

species (Triticum and Aegilops)

4

1.2 Composition of the wheat kernels: the endosperm, germ and bran 5

1.3 Alignment of amino acid sequences of GSP-1 from common wheat 8

1.4 Alignment of amino acid sequences of PINB-2v1 from common

wheat

10

1.5 Alignment of amino acid sequences of SINA and SINB in triticale 12

1.6 Alignment of the primary structure of PINA and PINB 14

1.7 Cysteine backbone and α-helix positioning in PINs and nsLTP 15

1.8 Structure prediction of PINA using I-TASSER 16

1.9 NMR solution structure of PuroA 17

1.10 Structure of PuroA in the presence of SDS (sodium dodecyl sulfate)

micelles

17

1.11 The predicted tertiary structures of PINA and mutants of PINA 18

1.12 Commonly cited models for antimicrobial peptide activity 26

2.1 Wheat and N. benthamiana seeds and plantlets 48

2.2 Diagram of indirect ELISA assay 61

2.3 Schematic showing the assembly of the three viral vector modules

and production of PIN proteins

64

2.4 The 3’module pICH11599 65

2.5 The other magnICON® viral vector modules 66

2.6 Generation of the Pins construct for magnICON® viral vectors 70

2.7 Agroinfiltration of N. benthamiana leaf 72

2.8 Principle of the BiFC assay 77

2.9 The pNBV (New BiFC Vector) modules 78

2.10 Plasmid map of pMLBART 80

2.11 Plasmid map of the P19 vector 81

2.12 Strategy used to construction of BiFC vectors for investigating

potential interactions of PIN proteins

84

2.13 Alignment of putative PINA and PINB 85

xvi

2.14 Generation of the mutant PINs construct for magnICON® viral

vectors

87

3.1 Second-round PCR of gene sections encoding mature PINs 91

3.2 Colony PCR for preliminary selection of clones 92

3.3 Example of double digests of clones in pICH11599 92

3.4 The 3’ module constructs encoding mature PIN with His-tag and

His/SEKDEL tags

93

3.5 N. benthamiana leaves 10 days post-infiltration 94

3.6 GFP production using two different targeting modules, in

N. benthamiana leaves (10 dpi)

95

3.7 Standard curve of BSA concentration for Bradford assay 96

3.8 Western blot analysis to determine GFP expression using anti-GFP

antibody

96

3.9 Coomassie Blue stained gel to analyse TSP from infiltrated leaves 97

3.10 Determination of PINA expression by western blotting of TSP

extracts from infiltrated leaves

98

3.11 Standard curve of 3% non-durum wheat dilutions for ELISA assay 99

3.12 Quantification of PINA proteins targeted to chloroplast, cytosol and

ER by ELISA

99

3.13 Coomassie Blue stained gel to analysis TSP from infiltrated leaves 100

3.14 Determination of PINB expressions by western blotting of TSP

extracts from infiltrated leaves

101

3.15 Quantification of PINB proteins targeted to chloroplast, cytosol and

ER by ELISA

102

3.16 Detection of PIN proteins harvested at different times post-

infiltration, by ELISA

102

3.17 Heat sensitivity of plant-expressed PINA and PINB detected by

western blot

103

3.18 Determination of PINA and PINB protein expression in infiltrated

leaves with viral vector module by western blot of TSP in expected

size

104

3.19 SDS-PAGE of TSP and wash steps of His-tag purification under

native conditions

105

xvii

3.20 SDS-PAGE and western blot analysis with Durotest antibody for

eluted fraction on Ni-NTA resin columns based on His-tag

purification under native conditions

106

3.21 SDS-PAGE and western blot analysis with Durotest antibody for

eluted fraction on columns with 6M urea in elution buffer

107

3.22 Fractions in the course of extraction procedure 108

3.23 SDS-PAGE for fractions eluted from the Butyl-Toyopearl column

based on hydrophobic interaction system

109

3.24 SDS-PAGE of eluted fraction on Butyl-Toyopearl column based on

hydrophobic interaction system and sodium phytate precipitation

109

3.25 Western blot of eluted fractions on Butyl-Toyopearl column based

on hydrophobic interaction system

110

3.26 Western blot analysis of PIN proteins purified with two different

systems

111

3.27 Example of Minimum Inhibitory Concentration (MIC) assay for

PuroA peptide and plant-made recombinant PIN proteins against

E. coli

113

3.28 Example of microdilution plate Minimum Inhibitory Concentration

(MIC) assay for filamentous fungi against R. solani

114

3.29 Haemolytic activity assays against sheep red blood cells (RBCs) 117

4.1 Second-round PCR of gene sections encoding mature PINs and

DNA plasmids of empty pNBV vectors

127

4.2 Colony PCR for preliminary selection of clones 128

4.3 The pNBV module constructs encoding mature PINA and PINB

proteins

129

4.4 Alignments sequence of PINA in pNBV construct 130

4.5 Alignments sequence of PINB in pNBV construct 131

4.6 BiFC visualisation in N. benthamiana leaves 133

4.7 BiFC visualisation of PINA-PINB interactions in N. benthamiana

leaves

134

4.8 BiFC visualisation of PINA-PINA and PINB-PINB interactions in

N. benthamiana leaves

135

4.9 Determination of interacting form of PINA+PINB and PINB+PINB 136

xviii

by western blot analysis from TSP extract

4.10 Native gel electrophoresis and UV imaging of YFP fluorescence of

PIN protein complexes

137

4.11 Amplification of inserts from PINA∆HD and PINB∆HD constructs 138

4.12 The pNBV module constructs generated encoding mutant PIN

proteins

139

4.13 Sequences of PINA∆HD in pNBV construct 139

4.14 Sequences of PINB∆HD in pNBV construct 140

4.15 Infiltrated mixtures of constructs into N. benthamiana leaves side by

side for detect and compare the interaction of wild type and mutant

form of PIN proteins

141

4.16 Western blot analysis to determine the interacting forms of wild

type and mutant PIN proteins

142

4.17 The pNBV module construct generated encoding PINA∆TRD

protein

143

4.18 Sequences of PINA∆TRD in pNBV construct 143

4.19 The 3’ module constructs encoding mutant PIN proteins with His-

tag

145

4.20 Sequences of PINA∆HD-His and PINA∆TRD-His in 3’ module

(pICH11599)

146

4.21 Sequences of PINB∆HD-His in 3’ module (pICH11599) 147

4.22 Determination of wild type and mutant PIN proteins expressions in

infiltrated leaves with chloroplast viral vector module by western

blotting of TSP extract

148

4.23 Structure prediction using PyMOL and prediction secondary

structure for PINA

150

4.24 Structure prediction using PyMOL and prediction secondary

structure for PINB

151

4.25 Modeling result of COTH for PINA and PINB Complex in

horizontal and vertical direction

153

4.26 Modeling result of COTH for PINA∆HD and PINB∆HD Complex 154

xix

List of Tables 1.1 The mutations identified in Pina-D1a and Pinb-D1a alleles in

common wheat (T. aestivum)

11

1.2 Reported recombinant PIN proteins 20

1.3 Reported antibacterial and antifungal activities of PIN proteins 23

1.4 Reported in vivo antimicrobial activity of PINs 24

1.5 Reported antibacterial and antifungal activities of PIN-based

peptides

25

1.6 Summary of factors affecting gene transcription, mRNA translation

and protein accumulation

31

1.7 AMPs heterologous produced in Nicotiana tabacum 38

2.1 Instruments and apparatus used 42

2.2 Commercial kits 43

2.3 Antibodies for western blot analysis 44

2.4 Composition of general buffers and solutions 44

2.5 Composition of protein purification buffers 46

2.6 Primer used for the wild type full length Pina-D1 and Pinb-D1

amplifications

51

2.7 Primers used in directional cloning into 3’ viral module, pICH11599 68

2.8 Genes cloned with His-tag and SEKDEL tag into pICH11599 68

2.9 Tri-partatite infiltration of magnICON® constructs into

N. benthamiana

71

2.10 Protein extraction buffers used in this study 73

2.11 Primers used in directional cloning into pNBV vectors 79

3.1 Quantification of TSP for targeted expression of GFP using

Bradford assay

96

3.2 ELISA data analysis for quantification of recombinant PINA

(rPINA) protein

99

3.3 ELISA data analysis for quantification of recombinant PINB

(rPINB) protein

101

3.4 Antibacterial activity of plant made recombinant PINA and PINB 113

3.5 Antifungal activity of plant made recombinant PINA and PINB 115

xx

3.6 Stability of recombinant PINs at different incubation temperature 116

3.7 Haemolytic activity of plant-made recombinant PINs 117

4.1 BiFC constructs for PIN proteins (wild type and mutant) to detect

fluorescence signal

144

CHAPTER 1

General introduction and literature review

Chapter 1 Introduction and literature review

- 2 -

1. General introduction

The information in this chapter provides an extensive review of the literature on

puroindoline (Pin) genes and puroindoline (PIN) proteins, their specific function in

controlling grain texture, as well as their possible biological roles as antimicrobial

proteins in wheat seeds.

Following the general overview, the introduction also highlights the importance of:

Understanding the roles of PIN proteins in lipid binding and impact on grain hardness

Identifying further PINs or PIN-based peptide structures

Plants as an alternative system for the expression of proteins in order to study the

structure and function of antimicrobial proteins and peptides

1.1 Introduction to wheat

Wheat (Triticum aestivum L.) is one of the most widely consumed grain crops in the

world. It is no doubt one of the most important cereal crops for humankind, with unique

end-use qualities that allow it to be processed into a range of flour-based foods such as

bread, biscuit and pastry products; additionally, it also has a role in the production of

starch and gluten (Li et al., 2012; Shewry, 2009). Wheat in the form of bread provides

essential nutrients like fibre, carbohydrates, protein, B vitamins, iron, calcium,

phosphorus, zinc, potassium and magnesium (Painter et al., 2002; Pena et al., 2002;

Wrigley, 2009). Wheat ranks third after maize (corn) and rice in terms of worldwide

production (Shewry, 2013).

Originating in the Middle East region about 10,000 years ago, wheat is the largest grain

crop based on area of cultivation (Dubcovsky and Dvorak, 2007; Matsuoka 2011;

Wrigley, 2009). Among agricultural crops, wheat is well adapted to a wide range of

soils, climates, and environmental conditions, unlike rice and maize that prefer tropical

environments (Gill et al., 2004). Durum wheat (Triticum turgidum L. var. durum) is

more adapted to the dry Mediterranean climate than bread wheat (Triticum aestivum L.)

(Shewry, 2009). Wheat varieties are categorised into six major classes based on

planting and harvesting dates, as well as hardness, shape and the colour of the kernels:

(1) hard red spring; (2) hard red winter; (3) soft red winter; (4) hard white wheat; (5)

soft white wheat; (6) durum (Rochelle, 2001).


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1.1.1 Evolution of wheat species

A detailed understanding for the origin of cultivated wheat has extended our knowledge

for improving it genetically. Wheat (genus Triticum) belongs to the Poaceae (or

Gramineae) family. The genus has three subclasses of ploidy: diploid (2n=2x=14),

tetraploid (2n=4x=28) and hexaploid (2n=6x=42) (Eckardt, 2001; Feldman, 1995;

Feldman, 2001). Three genomes, designated A, B and D, are involved in the formation

of the polyploidy series with seven pairs of chromosomes (Feldmann, 2001). Bread

wheat (Triticum aestivum), the most widely cultivated wheat today, is hexaploid

AABBDD and its origin has most likely occurred through one or more rare

hybridisation processes. Wild einkorn T. urartu (AA) crossed through natural

hybridisation with Aegilops speltoides (BB) to produce wild emmer T. turgidum L. ssp.

durum (AABB) (Chantret et al., 2005; Matsuoka, 2011). T. urartu is the diploid

progenitor of the A genome in tetraploid and hexaploid wheats (Haider, 2013). Another

hybridization between the tetraploid T. turgidum L. ssp. dicoccum (AABB) and the

diploid donor of the D genome, Ae. tauschii (2n =14, DD) restored the Hardness (Ha)

locus in T. aestivum (Chantret et al., 2005; Wrigley, 2009). The D genome is present in

T. aestivum but not in T. turgidum L. ssp. durum. Probably as a result of transposable

element insertions and two large deletions caused by illegitimate recombination, the Ha

locus in the D genome of hexaploid wheat (T. aestivum) is 29 kb smaller than in the D

genome of diploid Ae. tauschii (Chantret et al., 2005; Haider, 2013). Numerous studies

have attempted and published reports about the origin of the B genome in cultivated

wheat; however, the B genome donor remains uncertain. It is believed that the B

genome is closely related to the S genome from the Sitopsis group of the genus Ae.

speltoides (Feldman, 2001; Haider, 2013). The formation of tetraploid and hexaploid

wheat is summarised in Figure 1.1.

Figure 1.1 Schematic representation of the evolutionary history of wheat species (Triticum and Aegilops). Source: modified from Chantret et al. (2005) and Dubcovsky and Dvorak, (2007).


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1.1.2 Structure of the wheat grain

Wheat kernels consist of three distinct layers: the endosperm, germ and bran (Shewry,

2013) (Figure 1.2). The major part of wheat grain is endosperm and approximately

three-quarters of the endosperm includes starch, which has an important role in

determining the end-use of wheat grain. Differences in the endosperm texture of wheat

are an extremely important characteristic that have an effect on flour quality and yield

for human consumption. White flour contains the starchy endosperm, which contains a

high proportion of starch and gluten (Delcour and Hoseney, 2010; Turnbull and

Rahman, 2002).

Figure 1.2 Composition of the wheat kernels: the endosperm, germ and bran.

The germ is the embryo of the kernel and comprises only about 2.5% of the weight.

Wheat germ contains protein (~25%) and lipids (~8%), and is also an important source

of vitamins B and E (Cornell, 2003; Slavin, 2003). The bran layers surrounding the

endosperm are rich in vitamins and minerals and contains high-quality protein (19%), as

well as large amounts of insoluble dietary fibre (Wrigley et al., 2004). The bran layer

accounts for approximately 14.5% of the weight of the kernel. The aleurone layer is the

outermost layer of the endosperm and is located between the bran layer and endosperm.

The bran and aleurone layers play a role in protecting the embryo and nutrient-rich

endosperm against stress and pathogens (Gillies et al., 2012; Jerkovic et al., 2010).

Endosperm

Germ

Bran


- 6 -

1.2 Grain texture in wheat

Wheat hardness is a major quality trait and is defined as the force needed to crush the

kernels, which is required for the marketing and technological utilisation of wheat

(Morris, 2002), and also relates to the degree of hardness or softness of the grain. Grain

hardness is an essential factor, as it impacts milling and baking in the flour industry

(Bettge et al., 1995). Overall flour particle size, shape and flour density, starch damage,

water adsorption and milling yield are physical properties affected by hardness (Martin

et al., 2001). Grain hardness defines the quantitative variations within and across three

qualitative classes: ‘soft’ hexaploid wheats, ‘hard’ hexaploid wheats and the ‘very hard’

durum wheats. Moreover based on intermediate hardness, wheat has been classified as

‘medium hard’ and ‘medium soft’ (Morris, 2002). Bread wheat endosperm texture

varies from extremely soft to hard, while durum wheat has the hardest kernels of all

wheat species. In comparison with hard wheat, soft wheat generally yields flour with a

smaller average particle size and lower levels of damaged starch. Differences in

endosperm texture between hard and soft wheats are the most important factors for

functionality and marketing of wheat (Mikulikova, 2007). Generally, different types of

wheat are required by breeders, millers and bakers. Soft wheat flour with low protein

content is used to make cakes and pastries while hard wheat flour is suited for bread in

addition to other yeast raised products. Durum wheat with unique properties like high

protein content and gluten strength is used for pasta products and semolina (Douliez et

al., 2000; Morris, 2002).

Wheat endosperm hardness is controlled by genetic factors and biochemical factors

such as seed moisture, lipid and pentosan content. However, environmental conditions

also affect endosperm hardness (Konopka et al., 2005; Turnbull and Rahman et al.,

2002). Grain hardness has been linked to one major and several minor loci (Mattern et

al., 1973; Symes, 1965, 1969). The major locus was named Hardness (Ha) and shown

to be located at the distal end of the short arm of chromosome 5D (Doekes and

Belderok, 1976; Law et al., 1978; Mattern et al., 1973). Hard wheat has shown stronger

adhesion between starch granules and storage protein compared to soft wheat

(Simmonds et al., 1973). Studies conducted using scanning electron microscopy have

also revealed that more gluten adheres to the surface of starch granules isolated from


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hard wheat than to the surface isolated from soft wheat (Barlow et al., 1973; Hoseney

and Seib, 1973). It has been suggested that the degree of adhesion between starch and

gluten proteins is one of the essential factors for differences in endosperm hardness

between hard and soft wheat (Barlow et al., 1973; Hoseney and Seib, 1973; Simmonds

et al., 1973; Pauly et al., 2013).

1.2.1 Friabilin

Endosperm hardness is related to the occurrence of a 15 kDa protein complex called

‘friabilin’ on the surface of water-washed starch granules (Greenwell and Schofield,

1986). The protein was named ‘friabilin’ as it was observed that soft wheat was more

friable than hard wheat, while durum wheat lacked this protein (Morrison et al., 1992;

Morris, 2002). The amounts of starch granule associated protein from water washed

starch differ in soft and hard wheats (Jolly, 1993; Jolly et al., 1996; Rahman et al.,

1994). The occurrence of friabilin on the surface of water-washed starch granules

appears to be related to the level of bound polar lipids (glycolipids and phospholipids).

Polar lipids are present in high levels on the starch granule surface in soft wheat, but in

low levels in hard wheat (Capparelli et al., 2003; Greenblatt et al., 1995). Furthermore,

Greenblatt et al. (1995) showed that friabilin can be extracted with a propan-2-ol/water

(90:10) mixture, which is effective for removing starch-bound polar lipids. However,

with the same buffer, when no lipids are removed earlier, only a very small amount of

friabilin can be extracted. Friabilin components associated with starch granules through

polar lipids were shown to be involved in ionic and hydrophobic interactions

(Greenblatt et al., 1995). Friabilin protein analysis revealed that it is likely a mixture of

several polypeptides (Blochet et al., 1993; Morris et al., 1994; Oda and Schofield,

1997). The two major friabilin components are puroindoline-a (PINA) and

puroindoline-b (PINB) (Gautier et al., 1994). Grain Softness Protein-1 (GSP-1) and α-

amylase inhibitors (Rahman et al., 1994) are the minor components (Blochet et al.,

1993; Morris et al 1994). The GSP-1 protein (Figure 1.3) with approximately 164

amino acids long exhibits 40% identity and 60% similarity to the PINs with 10 cysteine

backbone and TRD with two tryptophan residues (Bhave and Morris, 2008a; Rahman et

al., 1994). Elmorjani et al. (2013) have shown that the GSP-1 protein may undergo

different proteolytic cleavages in the N and C-terminal regions of the pre-pro-protein.


- 8 -

Figure 1.3 GSP-1 putative protein sequence from common wheat. (T. aestivum) GenBank accession number: S48186

1.3 Puroindoline genes and grain texture

The Puroindoline (Pin) genes Pina-D1 and Pinb-D1, responsible for encoding the wild

type puroindoline proteins, are part of the Ha locus on the short arm of chromosome 5D

in common wheat (Gautier et al., 1994; Ragupathy and Cloutier, 2008). However,

during evolution and after polyploidisation of cultivated polyploid durum wheat, Pin

genes were deleted from chromosomes 5A and 5B. The Pin (Pina-D1, Pinb-D1) and

Gsp-1 genes are located in a 60 kbp DNA fragment of the Ha locus of Ae. tauschii,

which is the D-genome donor of T. aestivum (Pauly et al., 2013). Pin genes are located

only on the D-genome, while GSP-1 is present on all three wheat genomes (A, B and D)

in diploid, tetraploid and hexaploid wheat (Chantret et al., 2004, Chantret et al., 2005;

Morris 2002; Rahman et al., 1994). Without the D genome and thus both Pin genes and

also PIN proteins, resulting in the very hard durum wheats (Chantret et al., 2005).

1.3.1 Mutations, duplications and deletions in Pin genes

The Puroindoline genes (Pina-D1, Pinb-D1) contain 447 base pairs (bp) without introns

and both genes are 70.2% identical in the coding regions, but only 53% identical in the

3’-untranslated region (Gautier et al., 1994). The presence of both Pina-D1 and Pinb-

D1 genes in functional forms results in soft endosperm texture; however, mutations in,

or absence of either genes result in low levels of PIN protein(s) and hard endosperm

(Giroux and Morris 1998, Wall et al, 2010). The most common form of mutations are

single nucleotide polymorphisms (SNPs), which may result in an amino acid

substitution, or replacement with a stop codon, or a frame shift caused by a single base


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insertion or deletion (INDELs). The first report to show mutations in both Pin genes in

common wheats was by Chang et al. (2006). The mutations identified to date in Pina-

D1a and Pinb-D1a genes are listed in Table 1.1 (reviewed by Bhave and Morris, 2008a;

Nadolska-Orczyk et al., 2009).

1.3.2 Point mutations, duplications and deletions in Pina-D1 gene

The most common hardness-associated mutation to be reported in Pina-D1 was a null

mutation (Pina-D1b) and has a harder texture than other prevalent hardness alleles

(Giroux and Morris 1998; Morris, 2002. This results in the absence of Pina transcripts

and a lack of the PINA proteins in the kernel (Giroux and Morris 1998; Pauly et al.,

2013). Point mutations or SNPs have been reported in Pina-D1 genes (Chen et al.,

2006; Gazza et al., 2005; Massa et al., 2004). Chen et al. (2006) described different

SNPs in the mature protein sequence, which are: a) premature stop codon replacing

tryptophan at position 43 (Pina-D1n), b) proline to serine at position 35 (Pro35Ser)

(Pina-D1m). Mutations of serine in Pina-D1m may affect the lipid-binding ability of

the TRD due to the location of Ser in a loop between the first helix and the TRD (Bhave

and Morris, 2008a). Moreover, in Asian wheat cultivars, two types of deletion mutation

in Pina-D1 which results in PINA-null products have been reported. The 4.4kb deletion

mutant was designated Pina-D1r and the 10.5kb deletion was designated Pina-D1s

(Ikeda et al., 2010).

1.3.3 Point mutations, duplications and deletions in Pinb-D1 gene

Giroux and Morris (1997) were the first to report a single nucleotide change in Pinb-D1,

leading to the glycine-to-serine change at position 46 in PINB (G46S; Pinb-D1b), which

was associated with the hard texture in common wheat. This mutation has been

discovered widely in wheats around the world (Chen et al., 2006; Lillemo et al., 2006;

Pickering and Bhave, 2007; Tanaka et al. 2007; Xia et al., 2005). Wheats possessing

Pinb-D1b alleles were slightly softer than wheats with the Pina-D1b/Pinb-D1a (Morris,

2002). It has been suggested that this amino acid change affects lipid binding and starch

granule interaction, in addition to altering the tertiary structure of PINB at the TRD

(Bhave and Morris, 2008a). The point mutation of Pinb-D1c that causes a leucine-to-

proline change at position 60 (Leu60Pro) was first reported by Lillemo and Morris,


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(2000). Among the point mutations of Pinb-D1, mutations frequently occur at position

44 of the mature protein sequence, e.g., a tryptophan to arginine change at position 44

(Trp44Arg; Pinb-D1d) (Lillemo and Morris, 2000), tryptophan to early stop codon at

position 44 (Trp44stop codon; Pinb-D1f) (Morris et al., 2001), as well as a Pinb-D1q

(Trp44Leu) (Chen et al., 2005).

1.3.4 Pinb-2 genes

The six alleles of Pinb-2 gene have been reported and designated Pinb-2v1 to Pinb-2v6

(Chen et al., 2010a; Chen et al., 2010c; Ramalingam et al., 2012; Wilkinson et al.,

2008), which provide a potential new resource for minor variation in grain texture.

Mapping of the Pinb-2 genes in wheat reported Pinb-2v1 to be located on chromosome

7D in bread wheat and absent in durum wheat, Pinb-2v2 and Pinb-2v3 are located on

chromosome 7B and Pinb-2v4 is located on chromosome 7A (Chen et al., 2010a; Chen

et al., 2010c). The reported transcript levels of Pinb-2 variants suggest they are only

expressed at 10% the levels of the Pinb-D1 genes in common wheat (Wilkinson et al.,

2008). Additionally, RNA sequencing has shown that relative to Pinb, Pinb-2v1

expression was at 1%, and Pinb-2v2 and Pinb-2v3 was at ~8% of the expression levels

detected, while Pinb-2v4 was undetectable (Giroux et al., 2013). Pinb-2 genes appear

to have a greater impact on the grain texture of soft wheat than hard wheat and are

associated with increased grain texture in soft wheats (Chen et al., 2010b). Moreover,

the putative TRD of the PINB-2v1 proteins with two tryptophan residues (Figure 1.4)

has shown antimicrobial activity in vitro against a number of bacteria and fungi

(Ramalingam et al., 2012).

Figure 1.4 PINB-2v1 putative protein sequence. (T. aestivum) GenBank accession number: ADA7764

*SNP: Single nucleotide polymorphism *NS: Non-synonymous mutation, a change in the nucleotide sequence that does not lead to a change in the amino acid sequence

PIN allele Phenotype PIN protein Change to protein sequence* Reference Pina-D1a Soft Wild-type - Gautier et al. (1994) Pina-D1b Hard PINA null Gene deletion Giroux and Morris (1998) Pina-D1f Hard PINA Three SNPs. Arg58Gln and NS Ala19+Leu52 Massa et al. (2004) Pina-D1k Hard PINA null Gene deletion (Associated with Pinb deletion) Tranquilli et al. (2002)

Ikeda et al. (2005) Pina-D1l Hard PINA truncated Single base deletion, frame shift Gln61Lys, then a premature stop codon at pos120 Gazza et al. (2005) Pina-D1m Hard PINA Single SNP. Pro35Ser Chen et al. (2006) Pina-D1n Hard PINA Single SNP. Trp43stop, premature stop codon Chen et al. (2006) Pina-D1p Hard PINA truncated Single base deletion, frame shift Cys110Ala, then premature stop codon Chang et al. unpublished Pina-D1q Hard PINA Two SNP. Asn111Lys+lle112Leu Chang et al. (2006) Pina-D1r Hard PINA null Gene deletion Ikeda et al. (2010) Pina-D1s Hard PINA null Gene deletion Ikeda et al. (2010) Pina-D1t Hard PIN A truncated Single SNP. Trp41stop, premature stop codon Ramalingam et al. (2012) Pinb-D1a Soft Wild-type - Gautier et al. (1994) Pinb-D1b Hard PINB Single SNP. Gly46Ser Giroux and Morris (1997) Pinb-D1c Hard PINB Single SNP. Leu60Pro Lillemo and Morris (2000) Pinb-D1d Hard PINB Single SNP. Trp44Arg Lillemo and Morris (2000) Pinb-D1e Hard PINB truncated Single SNP.Trp39Stop,premature stop codon Morris et al. (2001) Pinb-D1f Hard PINB truncated Single SNP.Trp44stop,premature stop codon Morris et al. (2001) Pinb-D1g Hard PINB truncated Single SNP.Cys56stop,premature stop codon Morris et al. (2001) Pinb-D1l Hard PINB Single SNP.Lys45Glu Pan et al. (2004) Pinb-D1p Hard PINB truncated Single base deletion, frame shift Lys42Asn, then a premature stop at pos60 Chang et al. (2006) Pinb-D1q Hard PINB truncated Single SNP.Trp44Leu Ikeda et al. (2005) Xia et al.

(2005) Chen et al. (2005) Pinb-D1r Hard PINB truncated Single base deletion, frame shift Glu14Gly, then a premature stop at pos48 Ram et al. (2005) Pinb-D1s Hard PINB truncated Single base deletion and SNP, frame shift Glu14Gly, then a premature stop at pos48 Ram et al. (2005) Pinb-D1t Hard PINB Single SNP. Gly47Arg Chen et al. (2006) Pinb-D1u Hard PINB truncated Single base deletion, frame shift Glu14Ser, then a premature stop at pos18 Chen et al. (2007) Pinb-D1v Hard PINB Two SNPs. Ala8Thr+Leu9lle Chang et al. (2006) Pinb-D1w Hard PINB Single SNP.Ser115lle Chang et al. (2006) Pinb-D1aa Hard PINB truncated Single SNP. Single based deletion, frame shift Lys42Asn, a premature stop codon Li et al. (2008a) Pinb-D1ab Hard PINB truncated Single SNP. Gln99stop, premature stop codon Tanaka et al. (2008)

Table 1.1 The mutations identified in Pina-D1a and Pinb-D1a alleles in common wheat (T. aestivum)


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1.3.5 Pin-like genes in related species

Genes encoding puroindoline-like proteins do not occur in rice, maize or sorghum, but

are present in cereals related to wheat such as rye, barley and oat (Gautier et al., 2000;

Pauly et al., 2013). The PIN homologues in rye (Secale cereal L.) and triticale cultivar

are named secaloindoline a (SINA) and secaloindoline b (SINB) (Li et al., 2006;

Simeone and Lafiandra, 2005) (Figure 1.5). Until now, no relationship between

secaloindoline alleles and grain hardness has been reported in rye since rye cultivars

have generally soft endosperm texture with very low variation in kernel hardness

(Simeone and Lafiandra, 2005). Recently, Gasparis et al. (2013) reported RNAi-based

silencing of Sin genes resulted in a significant decrease in the level of transcripts and the

yield of both secaloindoline proteins but did not affect grain hardness in triticale cultivar

Wanad. Furthermore, barley (Hordeum vulgare L.) contains the PIN homologues

hordoindoline a (HINA) and hordoindoline b (HINB) and have been mapped on the

short arm of chromosome 5H (Beecher et al., 2002b). The Ha locus in barley has been

shown to be associated with variations in endosperm texture (Beecher et al., 2002b). In

addition, PIN homologues have been identified in oat (Avena sativa L.), designated

avenoindoline a and avenoindoline b (Gautier et al., 2000). Immunolocalisation studies

using Durotest antifriabilin antibody have detected tryptophanins (TRPs) proteins in oat

seed associated with the surface of starch granules (Mohammadi et al., 2007).

Figure 1.5 Alignment of amino acid sequences of SINA and SINB in triticale. GenBank accession numbers: AGO65289.1 and AGO65290.1 for SINA and SINB respectively


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1.4 Biochemical properties and structure of PIN proteins

N-terminal sequencing identified the basic cysteine-rich proteins Puroindoline-a (PINA)

and Puroindoline-b (PINB) as the major components of friabilin (Blochet et al., 1993).

The name ‘puroindoline’ is derived from the Greek word ‘puros’ for wheat, and

‘indoline’, referring to the indoline ring of the tryptophan-rich domain (Gautier et al.

1994). The PINs and other proteins (for instance storage proteins and the enzyme

granule-bound starch synthase I) with affinity for defatted starch granules were also

isolated from wheat endosperm using the nonionic detergent Triton X-114 (Blochet et

al., 1993; Bako et al., 2007).

PINA and PINB contain tryptophan residues in the unique tryptophan-rich domain

(TRD) and a highly conserved cysteine-rich backbone of 10 cysteine residues (Blochet

et al., 1993). The TRD of PINA consists of five tryptophan residues (WRWWKWWK;

position without the putative signal paptide: 38 to 45), while in PINB, it consists of

three (WPTKWWK; position without the putative signal paptide: 39 to 45) (Blochet et

al., 1993; Gautier et al., 1994). The full length PIN proteins consist of 148 amino acids

with a similar molecular mass of 13 kDa (Gautier et al., 1994). PIN proteins are highly

basic, with a calculated isoelectric point (pI) of 10.5 for PINA and 10.7 for PINB and

show 55% similarity at the amino acid level (Gautier et al., 1994). Both PINs are

synthesized as pre-pro-proteins containing an N-terminal signal peptide, which

comprises the first 28 amino acid residues for PINA and the first 29 amino acid

residues

for PINB (Gautier et al., 1994) (Figure 1.6). These N-terminal signal peptides may have

a role in intracellular targeting (Bhave and Morris, 2008a; Gautier et al., 1994). C-

terminal processing has also been suggested for both PINs and can result from slightly

different post-translation processing pathways (Day et al., 2006). Raman spectroscopy

studies have shown that PINA and PINB have a similar secondary structure at pH 7.

Both PINs consist of approximately 30% α-helices, 30% ß-sheets and 40% unordered

structures (Le Bihan et al., 1996). Far-UV circular dichroism (CD) measurements have

confirmed similarity between PINs in secondary structure (Kooijman et al., 1997).


- 14 -

The 10 cysteine residues form five intramolecular disulphide bonds. Disulphide bonds

are essential for stabilizing the α-helical structure and to maintain the native structure

and solubility of PINA and PINB (Le Bihan et al., 1996). Eight of the cysteines form a

particular pattern known as the “eight cysteine motif” (8CM), which appears widely

among plant proteins and has a similar tertiary structure, including four α-helical

structures (Figure 1.6) and variable loops (Pauly et al., 2013). Proteins containing this

motif show a wide range of functions in storage, enzyme inhibition, lipid transfer, plant

defence and cell wall structure (José-Estanyol et al., 2004). The cysteine residues may

have an important role in forming a network of disulphide bridges, which are important

for the maintenance of the three-dimensional (3D) structure of the molecule, together

with the central helical core (José-Estanyol et al., 2004). The TRD is located between

two additional cysteine residues. Kooijman et al. (1997) suggested that the TRD of

PINs has a role in the stability of the structure. The TRD confers hydrophobicity for

strong affinity in PIN interactions with lipids (Douliez et al., 2000; Kooijman et al.,

1997).

Figure 1.6 Alignment of the primary structure of PINA and PINB. Signal peptide in both PINs is highlighted before potential N-terminal cleavage sites (after the 28th

residue for PINA and after the 29th

residue for PINB). The 4-alpha helices are as

determined by Le Bihan et al. (1996) with the following locations, respectively for PINA and PINB: residues 17-28 and 18-29 for H1, 49-62 and 50-63 for H2, 69-76 and 70-77 for H3, and 87-100 and 89-101 for H4.


- 15 -

The high resolution structure of PINs has some limitations due to the strong aggregative

properties at high protein concentration and difficulties in obtaining a stable non-

aggregated solution required for crystallisation and NMR characterisation (Clifton et al.,

2011a; Marion et al., 2007). The 3D structure of PINs has not yet been determined.

However, a considerable amount of literature has been published on predicted models.

The first of these was based on the folding pattern of the eight-cysteine motif in

nonspecific lipid transfer proteins (ns-LTP). The eight-cysteine motif, including the

Cys-Cys and Cys-arginine-Cys characteristic of both PINs, is also found in nsLTPs.

The similarities of the disulphide bonds support the suggestion that nsLTPs and

puroindolines are related in their primary and secondary structure, and display similar

folding properties (Douliez et al., 2000; Marion et al., 2007) (Figure 1.7). PINs have

five disulphide bonds while ns-LTPs have four. The TRD of PINs is located in a loop

between the first and second α-helix, outside the protein based on the 3D model for ns-

LTP (Doulies et al., 2000; Kooijman et al., 1997; Marion et al., 1994). In addition, the

variable loops connected to the α-helices are the functional regions of the eight-cysteine

motif proteins (José-Estanyol et al., 2004), which seems appropriate for PINs. There is

also a unique tyrosine residue (positions without the putative signal paptide: 23 and 24

for PINA and PINB respectively) in the first α-helix, which may be functionally

important. Tyrosine is not bound to a negatively charged carboxylate of the protein, but

is hydrogen-bonded to water molecules (Le Bihan et al., 1996).

Figure 1.7 Cysteine backbone and α-helix positioning in PINs and nsLTP. Source: Douliez et al. (2000)


- 16 -

Recently, a different 3D structure model of PINA using iterative threading assembly

refinement (I-TASSER) has been revealed for structure prediction by Lesage et al.

(2011) (Figure 1.8). The prediction result for PINA displayed the five disulphide

bonds: Cys20/Cys55 and Cys56/Cys104 are connected and readily form disulphide

bonds; Cys11/Cys66 and Cys68/Cys110 are positioned at close proximity (Lesage et al.

2011) and the TRD is an extension stabilised by a Cys28/Cys48 disulphide bridge in

PINA and a Cys29/Cys48 disulphide bridge in PINB (Le Bihan et al., 1996). PINs have

several features of membrane proteins, such as high levels of α-helices. In addition,

both PIN proteins can be extracted with non-ionic detergent like TX-114; however,

none of them contains a typical trans-membrane region. Moreover, after extraction in

the absence of detergents, puroindolines are fairly water-soluble, and act differently

from membrane proteins (Lesage et al., 2011).

Figure 1.8 Structure prediction of PINA using I-TASSER. Source: Lesage et al. (2011)

PINA forms aggregate under acidic and high ionic strength conditions and at low

temperatures, where the TRD plays an important role in the aggregate formation (Le

Bihan et al., 1996). Clifton et al. (2011a) confirmed this observation and has shown that

PINA forms monodisperse prolate ellipsoidal micelles in a solution consisting of 38

PINA molecules and is stable in a wide range of pH and temperatures. Self-assembly of

PINA is suggested to be driven by intermolecular hydrophobic forces between the

tryptophan at the TRD. Although PINB shows more than 50% amino acid similarity

with PINA, no self-assembly into micelles has been reported. This may be due to the

fewer tryptophan residues in the PINB TRD, resulting in weaker intermolecular

hydrophobic interactions (Clifton et al., 2011a). The structure of a PINA section, i.e.,


- 17 -

the peptide PuroA (FPVTWRWWKWWKG) was determined by NMR (Jing et al.,

2003) (Figure 1.9) and showed that all positively charged residues are located close to

the tryptophan indole rings (Jing et al., 2003). Furthermore, the 3D structure of micelle-

bound PuroA shows that all positively charged residues are placed close to the

tryptophan indole rings, which have a role in cation-interactions and allow PuroA to

penetrate and disrupt bacterial membrane (Chan et al., 2006) (Figure 1.10).

Figure 1.9 NMR solution structure of PuroA. Source: Jing et al. (2003). 13 amino acids corresponding to the tryptophan-rich domain (TRD) of puroindoline a. tryptophan (W), arginine (R) and lysine (K) residues are numbered based on their position in the PuroA peptide.

Figure 1.10 Structure of PuroA in the presence of SDS (sodium dodecyl sulfate) micelles. Source: Chan et al. (2006). The cation–π arrangement between tryptophan and arginine residues on the left side of the peptide are shown; tryptophan residues are highlighted in green, phenylalanine is show red, and lysine and arginine residues are presented in blue colour. The disulphide bridges are shown in yellow stick representation.


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A prediction of the tertiary structure of wild type PINA (represented as ABC) and

mutants of PINA (contain two (ABBC) or three (ABBBC) copies of TRD) proteins was

obtained using the program Protein Homology/analogue Y Recognition Engine v2.0

(Phyre2) (Miao et al., 2012) (Figure 1.11). Furthermore, the secondary structures of the

purified recombinant proteins were estimated by Circular dichroism (CD) spectroscopy

(Miao et al, 2012).

Figure 1.11 The predicted tertiary structures of PINA and mutants of PINA. Source: Miao et al. (2012). a and d: Wild type PINA (ABC); b and e: mutant of PINA (ABBC); c and f: mutant of PINA (ABBBC). TRD(s) are shown in boxes; arrows point to the hydrophobic tryptophan residues (in red).

1.4.1 Heterologous expression of recombinant PIN proteins

Heterologous expression of PINs is an important strategy for scaling up their production

and functional analysis, and has been described by several authors (Capparelli et al.,

2006; Capparelli et al., 2007; Issaly et al., 2001; Miao et al., 2012; Sorrentino et al.,

2009). The yeast Pichia pastoris has been successfully used to express recombinant

PINA protein (Issaly et al., 2001). During fermentation the addition of Triton X-114

increased the production yield of the recombinant PINA by 10-fold to 14 mg/L (of

which 80% was soluble), compared to concentrations of recombinant PINA previously

detected without Triton X-114 (Issaly et al., 2001).

The cysteine-rich PIN proteins fused with an N-terminal His-tag (6 histidine amino

acids) were successfully expressed in Escherichia coli strains BL21 and BL21pLysS as


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18 kDa proteins (Capparelli et al., 2006). Both types of cells expressed His-PINA and

His-PINB in the insoluble fraction, while His-PINs did not refold properly. His-protein

purification using Ni-NTA spin column under denaturing conditions allowed the

proteins to be obtained in purified form. Additionally, both PINs were expressed in E.

coli strain DH5α with a GST-tag (Glutathione-S-Transferase, known to increase the

solubility of the target protein) as 38 kDa proteins and correctly folded. Further, only

recombinant GST-PINs could be efficiently purified, refolded and cleaved from their

tag (Capparelli et al., 2006).

In summary, 1.5 mg recombinant His and GST tagged PINs were obtained from a 1 L

culture (Capparelli et al., 2006). In addition, Capparelli et al. (2007) described the

expression of PINA and PINB in the Origami strain of E. coli, with yields of the

purified proteins at 1.8 and 0.65 mg per litre of culture, respectively, the yield being 2.6

mg per litre of culture for each protein before cleaving the tag. Wild type PINA (ABC)

and mutants of PINA (with two (ABBC) or three (ABBBC) copies of TRD) were

expressed in E. coli strain Rosetta-gami (DE3), with a NusA fusion tag at the N-

terminal to promote the solubility. The strain Rosetta-gami (DE3) has trxB and gor

mutations, which enhance disulphide bond formation in the E. coli cytoplasm and

provides rare codon tRNAs, hence it can be assumed that the recombinant PINs

expressed in it were folded correctly. A low temperature pre-culture and induction

strategy was also used to substantially increase the yield of soluble, functional PINs

(Miao et al., 2012). Alongside the use of classical organism systems such as E. coli and

yeast to express recombinant PIN proteins, molecular farming approaches with plant

system have also been used. The mature sequence of PINs (lacking the signal peptide)

was fused with an N-terminal 3×FLAG tag (DYKDDDDK) and sub-cloned into a plant

expression vector to target both PINs in the apoplast and the PINB in the chloroplast of

Nicotiana tabacum cv. Bright Yellow 2 (BY-2) cells. PINB targeted to the chloroplast

showed a 20 kDa band based on immunoblot analysis using the MαFLAG-M2

monoclonal antibody and yields of 0.35% of the total soluble proteins. Nevertheless, in

stable suspension-cell cultures, no positive signals were observed for PINA and PINB

targeted to the apoplast in either the cell protein extract or the culture medium

(Sorrentino et al., 2009). The reported recombinant PIN proteins are listed in Table 1.2.


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Table 1.2 Reported recombinant PIN proteins Protein Host Result References

PINA and PINB

Escherichia coli (BL21, BL21pLysS and DH5α)

Obtained His tagged and GST tagged PINs, 1.5 mg recombinant protein from 1 l culture

Capparelli et al. (2006)

Escherichia coli (Origami)

Obtained 1.8 mg l−1 PINA and 0.65 mg l−1

PINB yields of purified protein Capparelli et al. (2007)

PINA

Yeast (Pichia pastoris) Obtained 80% of soluble recombinant PINA Issaly et al.

(2001)

Escherichia coli (Rosetta-gami, DE3)

Expressed and purified PINA and two artificial mutants of PINA

Miao et al. (2012)

PINB Tobacco (Nicotiana tabacum) Expressed recombinant PINB; 0.35% of TSP Sorrentino et al.

(2009)

1.4.2 Evidence of transgenic Pin genes altering grain texture

PINs may be synthesised in the aleurone and transported to the endosperm (Gautier et

al., 1994). PIN localisation in wheat indicated that both proteins were co-localised in

the aleurone layer and the starchy endosperm of rehydrated mature seeds, however,

neither PIN could be found in root, leaf, shoot or hypocotyls of seedlings (Capparelli et

al., 2005; Digeon et al., 1999; Dubreil et al., 1998). Additionally, immunofluorescent

localisation study confirmed the presence of both PINs at the starch granule surface

(Feiz et al., 2009c). In transgenic wheat, both Pin genes were expressed in the starch

endosperm cells (Wiley et al., 2007). However, PINs have been found in protein bodies

during endosperm development by Lesage et al. (2011). It is currently hypothesised

that PINs determine wheat hardness by stabilising the amyloplast membrane on the

surface of starch granules during grain desiccation, thereby preventing total breakdown

of the lipids when the wheat kernel ripens resulting in soft genotypes containing more

phospholipids and glycolipids than hard textured genotypes (Feiz et al., 2009b; Kim et

al., 2012b).

Expression of both wild type Pin alleles (Pina-D1a and Pinb-D1a) in rice, under the

control of the maize ubiquitin promoter, resulted in reduced hardness of the transgenic

rice seeds texture and decreased levels of damaged starch granules, as well as the

average particle size during milling (Krishnamurthy and Giroux, 2001). A softer texture

in transgenic Japonica rice was also observed following Pinb-D1a gene expression

(Wada et al., 2010). Two different transgenic rice lines were also used for

histochemical analysis of the endosperm cell and immunodetection of wheat PINA and


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PINB using the Durotest anti-friabilin antibody and the results suggest that they were

localised between starch granules in the rice endosperm cell (Fujiwara et al., 2014). In

transgenic corn, the expression of Pin genes (Pina-D1a and Pinb-D1a), resulted in an

increase in germ size, germ yield and seed oil content (Zhang et al., 2010a).

The expression of wild type Pinb-D1a in the hard wheat cv. Hi-Line resulted in soft

phenotype, increased friabilin yield and decreased damaged starch (Beecher et al.,

2002a). Further, in a similar study by Hogg et al. (2004), the transgenic hard wheat

cultivar ‘Hi-Line’ with wild type Pina-D1a, Pinb-D1a or both showed reduced softness;

however, expression of PINB resulted in softer kernel. Swan et al. (2006a) reported that

the crossing of the transgenic ‘Hi-Line’ with a soft wheat resulted in progenies

expressing different levels of PINs; those expressing PINB were softer and had more

starch-bound PINs compared to those expressing PINA which only had an increase of

starch-bound PINA. This work supports the need for both PINs in their wild form for

the soft endosperm texture and may indicate that PINB has a role in assisting PINA

binding to starch.

The over-expression of foreign Pina has been associated with co-suppression of the

endogenous Pina (silencing of Pina), resulting in significantly harder kernels (Xia et al.,

2008). The silencing of either Pin was found to decrease the expression of the other Pin

genes and increased grain hardness, based on RNA interference (RNAi) studies in

transgenic wheat (Gasparis et al., 2011). The introduction of Pina-D1a and Pinb-D1a

(Gazza et al., 2008) or Pina-D1a (Li et al., 2014) into durum wheat significantly

reduced its hardness value. Pin genes from soft wheat cv. Chinese Spring were

transferred through ph1b-mediated homoeologous recombination into durum wheat, and

these durum lines were stable and could be used in crossing the soft endosperm texture

to other durum wheat cultivars (Morris et al., 2011).


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1.5 Evidence of in vitro antimicrobial activity of PINs

PIN proteins are located in the starchy endosperm and the aleurone cells of wheat seed,

and have a possible biological role as antimicrobial proteins. Therefore, a considerable

number of studies have been published on the in vitro antibacterial and antifungal

activities of PIN proteins (Capparelli et al., 2005; Capparelli et al., 2006; Capparelli et

al., 2007; Dubreil et al., 1998; Jing et al., 2003). However, the biological function of

PINs is not yet clear. PINA and PINB purified from wheat display in vitro antifungal

activity against Alternaria brassicola, Ascochyta pisi, Botrytis cineria, Fusarium

culmorum and Verticillium dahlia, with the antifungal activity of PINB higher than that

of PINA (Dubreil et al., 1998) (Table 1.3). Both wheat PINs show similar antibacterial

activity and it appears that synergy between the two proteins may enhance their

antimicrobial activity (Capparelli et al., 2005). Further, recombinant PINs expressed

and purified from bacterial expression systems (i.e., E. coli) folded similarly to the

native proteins purified from wheat and revealed the same extent of in vitro antibacterial

activity (Capparelli et al., 2006) (Table 1.3). Antimicrobial activity against E. coli (91%

growth inhibition) has been demonstrated for chloroplast targeted PINB in Nicotiana

tabacum cells (Sorrentino et al., 2009). Mutant forms of recombinant PINA that contain

an extra copy of the TRD have shown higher antibacterial activities (70 µg/mL against

E. coli) than the wild type PINA (90 µg/mL against E. coli) (Miao et al., 2012),

providing evidence that the TRD has a role in their antimicrobial activity. Other studies

have also proposed the TRD of PINs to have a role for their antibacterial and antifungal

activities (Evrard et al., 2008; Jing et al., 2003; Phillips et al., 2011). The PIN proteins

also seem to be a promising tool in topical antibiotics for bacterial infections.

Recombinant PINs have been shown to be effective in ectopic treatments for fungal skin

infections caused by Staphylococcus epidermidis without exhibiting any haemolytic

activity or toxicity to mouse macrophage cells in vitro, and ability to kill intracellular S.

epidermidis (Capparelli et al., 2007). Furthermore, Palumbo et al. (2010) showed

potential antibacterial activity against Listeria monocytogenes in a mouse model with

intravenously injected PIN proteins.


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Table 1.3 Reported antibacterial and antifungal activities of PIN proteins

Puroindoline proteins purified from wheat

Protein Organism Antimicrobial activity (µg/mL) Reference

PINA Alternaria brassicicola Ascochyta pisi Fusarium culmorum Verticillium dahliae

100 200 100 100

Dubreil et al. (1998)

Escherichia coli Staphylococcus aureus Agrobacterium tumefaciens Pseudomonas syringae Erwinia carotovora Clavibacter michiganensis

30 30 35 50 50 50


PINB Alternaria brassicicola Ascochyta pisi Fusarium culmorum Verticillium dahliae

20 70 40 30

Dubreil et al. (1998)

Escherichia coli Staphylococcus aureus Agrobacterium tumefaciens Pseudomonas syringae Erwinia carotovora Clavibacter michiganensis

30 30 35 50 50 50


Puroindoline proteins expressed in bacterial cells

Protein Organism Antimicrobial activity (µg/mL) Reference

PINA Escherichia coli Staphylococcus aureus

30 30


Staphylococcus epidermidis 30 Capparelli et al. (2007) Escherichia coli Staphylococcus aureus

90 150

Miao et al. (2012)

PINB Escherichia coli Staphylococcus aureus

30 30


Staphylococcus epidermidis 30 Capparelli et al. (2007) Puroindoline protein expressed in Nicotiana tabacum Protein Organism Percentage growth inhibition Reference PINB Escherichia coli 91% Sorrentino et al.(2009)

1.5.1 Evidence of in vivo antimicrobial activity of PINs

PINs have been shown to induce a significant increase in plant resistance towards

microbial pathogens by over-expression and can therefore be applied in the preservation

of food products. However, no in vivo work had been reported until 2001, when the

earliest evidence of in vivo antimicrobial activity for PIN expression was reported.

Transgenic rice expressing PINA and/or PINB showed in vivo antimicrobial activity and

higher resistance against two major fungal pathogens in rice, Magnaportha grisea and

Rhizoctonia solani, which are causal agents of rice blast and sheath blight, respectively

(Krishnamurthy et al., 2001).


- 24 -

The durum wheat varieties ‘Luna’ and ‘Venusia’ which naturally lack Pina and Pinb

genes were transformed with the Pina-D1a gene, under the control of the maize

ubiquitin promoter, and both varieties showed significantly increased resistance to

Puccinia triticina (leaf rust fungus) and an increase in harvest yield (Luo et al., 2008).

Kim et al. (2012a) compared seed fungal resistance by overexpression of PINA, PINB

or both PINs in wheat to near-isogenic lines (NILs) with mutations in PINA or PINB

and found that lines expressing both PINs had reduced Penicillium sp. fungal infection

in seeds and increased germination. Expressing both Pin genes into corn was also

effective in reducing the symptoms of Cochliobolus heterostrophus, the corn southern

leaf blight pathogen, by 42% (Zhang et al., 2011).

PINs can also be expressed successfully in dicots. For example, two apple genotypes

transformed via Agrobacterium tumefaciens with Pinb-D1a under the control of the

cauliflower mosaic virus 35S promoter (CaMV35S) led to PINB yield to 0.24% of the

total soluble proteins. In addition, the expression of Pinb alone or in association with a

natural resistance gene Vf, induced partial resistance to scab, a fungal disease caused by

Venturia inaqualis (Faize et al., 2004). The in vitro antifungal activity of PINB against

V. inaqualis had previously been shown by Chevreau et al. (2001). The reported in vivo

antimicrobial activity of PINs is listed in Table 1.4.

Table 1.4 Reported in vivo antimicrobial activity of PINs

Gene Host Resistance against Disease Reference

Pina and/or Pinb

Transgenic rice Magnaportha grisea

and Rhizoctonia solani

rice blast and

sheath blight

Krishnamurthy et al. (2001)

Transgenic wheat lines Penicillium sp. fungal infection

in seeds Kim et al. (2012a)

Pina and Pinb

Transgenic corn Cochliobolus heterostrophus

southern corn leaf blight Zhang et al. (2011)

Pina Transgenic tetraploid wheat Puccinia triticina leaf rust Luo et al. (2008)

Pinb Transgenic apple lines Venturia inaqualis apple scab Faize et al. (2004)


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1.5.2 Evidence of antimicrobial activity of PIN-based peptides

Several studies have utilised chemically synthesised peptides, which have been

designated to identify the bioactive domain of the PIN proteins. The PuroA

(FPVTWRWWKWWKG-NH2) and PuroB (FPVTWPTKWWKG-NH2) peptides were

designed based on the TRD of PINA and PINB, respectively, to identify the

antimicrobial activity. PuroA showed a bactericidal effect on both Gram positive and

Gram negative bacteria; in contrast, PuroB showed no activity even at the highest

concentration tested (Jing et al., 2003). Wild type and variant forms of synthetic

peptide were tested against bacteria, as well as phyto-pathogenic fungi (Table 1.5).

PuroB showed specific activity against Rhizoctonia sp., with minimum inhibitory

concentrations (MIC) 64 µg/mL, however, PuroA had a much higher antibacterial

activity than PuroB. The number of tryptophan and positively charged residues can be a

possible explanation for these different functions (Jing et al., 2003; Phillips et al., 2011).

Evrard et al. (2008) showed that two tryptophan residues (W41 and W44) in the TRD

were essential for PINA to interact with the membrane of Saccharomyces cerevisiae,

while the lysine residues (K42 and K45) in the TRD of PINB seem to be more

important for yeast membrane interaction.

Table 1.5 Reported antibacterial and antifungal activities of PIN-based peptides

Name Sequence Organism Activity (µg/mL) References

PuroA

FPVTWRWWKWWKG-NH2

Escherichia coli Staphylococcus aureus

14 33

Jing et al. (2003)

Escherichia coli Staphylococcus aureus Collectotrichum graminicola Drechslera brizae Rhizoctonia cerealis Rhizoctonia solani

16 16 250 250 64 32

Phillips et al. (2011)

PuroB FPVTWPTKWWKG-NH2 Rhizoctonia cerealis Rhizoctonia solani

64 64

Phillips et al. (2011)

1.5.3 Possible mode of action for PINs as antimicrobial proteins and peptides

Anti-microbial peptides or proteins (AMPs) identified from prokaryotes and eukaryotes

are usually less than 100 amino acid residues long and often have a high frequency of

positively charged amino acids (Silva et al., 2011). They are strongly cationic (pI: 8.9-

10.7) having a net charge of 2+ to 7+, mainly due to the presence of arginine, lysine and


- 26 -

histidine residues, heat-stable (100°C, 15 minutes), and additionally, they have may

have 50% hydrophobic amino acids and do not have any lysing/killing effects on

eukaryotic cells (Hancock and Chapple 1999; Li et al., 2012b).

AMPs have been shown to be an important and ancient mechanism of natural resistance,

a rapid and metabolically inexpensive first line of defence against pathogens (Brokaert

et al., 1997; Egorov et al., 2005). The antimicrobial activity of such peptides was

observed to involve ionic interactions between their basic residues and the negatively

charged bacterial membrane constituents, such as phospholipid head groups (Jenssen et

al. 2006; Melo et al., 2009; Shai, 2002). On the other hand, the outermost layer of the

eukaryotic membranes is composed of lipids with no net charge and zwitterionic

headgroups positioned within the inner layers; membrane-binding is controlled by

hydrophobic rather than ionic interactions (Lee et al., 2011a). The simplest models of

microbe permeation by antimicrobial peptides involve the formation of transbilayer

pores or channels, as shown by the models in Figure 1.12 (Wimley, 2010).

Figure 1.12 Commonly cited models for antimicrobial peptide activity. Source: Wimley (2010).


- 27 -

During the past 15 years, a wide range of AMPs have been shown to play essential roles

in plant defence systems. Their sizes range from 2 to 9 kDa and many of them have a

cysteine-rich structure with 6 or 8 cysteine residues involved in disulphide bridges; e.g.,

thionins, defensins, non-specific lipid transfer proteins (nsLTPs), hevein- and knottin-

like peptides, MBP1, IbAMP and snakins (Broekaert et al., 1997; Benko-Iseppon et al.,

2010; Egorov et al., 2005; García-Olmedo et al., 1998; Nawrot et al., 2014; Odintsova et

al., 2009). Some of the cysteine-rich AMPs, such as defensins, thionins and LTPs, are

observed in wheat plants (Egorov et al., 2005). The hevein-like peptide with a 10

cysteine motif isolated from Triticum kiharae has been shown to have antifungal and

antibacterial activity (Dubovskii et al., 2011; Odintsova et al., 2009).

The wheat puroindoline proteins share properties with other AMPs, e.g., the low

molecular weight and the disulphide bond structure (Chan et al., 2006; Schibli et al.,

2002). Dubreil et al. (1997) showed that PINA is able to interact tightly with both

wheat phospholipids and glycolipids while PINB interacts only with negatively charged

phospholipids. However, the integrity of the highly hydrophobic TRD is required for

interaction with neutral polar lipids. Using fluorescence emission and circular

dichroism (CD) spectroscopy the involvement of the TRD region in lipid binding

confers strong affinity for polar lipids to PINs (Kooijman et al., 1997). The TRD in

PINA is involved in insertion into the lipid biofilm and interacts with the lipid head

group region of anionic phospholipids, mostly due to external hydrophobic residues and

cationic side chains at the TRD (Kooijman et al., 1998). The involvement of tryptophan

and basic residues in the TRD of PINA has been proposed as important for lipid binding

and its insertion into membranes (Kooijman et al., 1997; Le Guerneve et al., 1998).

Tryptophan-rich antimicrobial peptides have shown unique amphipathic properties from

the side chain of tryptophan (Schiffer et al., 1992).

The in vitro antimicrobial properties of cationic peptides based on the TRD of the PINA

and/or PINB proteins (Capparelli et al., 2005; Charnet et al., 2003; Jing et al., 2003;

Phillips et al., 2011; Ramalingam et al., 2012) have been summarized above. PuroA

binds strongly with and penetrates deeper into negatively charged phospholipid head

groups of microbial membranes and disrupts the membrane bilayer structure by forming


- 28 -

cation interactions between arginine and lysine and the indole side chains of tryptophan

(Jing et al., 2003). Tryptophan and arginine residues play a key role in the antimicrobial

action of peptides such as tritrpticin and indolicidin (Chan et al., 2006). However, it has

been suggested that the position rather than the number of tryptophans is an important

factor (Bi et al., 2013). Membrane interaction studies in yeast have shown that

tryptophan-41 and tryptophan-44 at the TRD of PINA may be essential for interaction

with fungal plasma membranes. However, for PINB, none of the tryptophan residues

were required, whereas the positively charged lysine-42 and lysine-45 were essential for

yeast membrane interaction of PINB (Evrard et al., 2008). In addition, single

substitution at the TRD of PINB, of a glycine to a more polar serine residue (Gly46Ser)

and a tryptophan to a basic arginine residue (Trp44Arg) decreased the selectivity

towards anionic phospholipids (Clifton et al., 2007a; Clifton et al., 2007b), which

supports the hypothesis that tryptophan may influence membrane selectivity. Further,

Phillips et al. (2011) showed the antimicrobial properties of cationic peptides based on

the unique TRD of the PIN proteins. They have shown reduced activity of PINA

peptide upon substitution of tryptophan residues with phenylalanine within the TRD.

For antifungal activity of the PINB-TRD, lysine-45 and tryptophan-39 have indicated

important roles, while tryptophan-44 does not appear to be essential and substitution of

lysin-45 to glutamic acid in Pinb-L abolished any observable antifungal activity

(Phillips et al., 2011). Microbial membrane interaction via the polar lipids and

membranes may be required as a mode of action of the PIN proteins (Blochet et al.

1993). Using electrophysiological experiments on Xenopus oocytes and artificial planar

bilayers, channel formation mechanisms were shown to be important for puroindolines

antimicrobial activity (Charnet et al., 2003). Moreover, the PIN proteins appear to act

synergistically in combating pathogens not only with α-purothionins but also with one

another (Capparelli et al., 2005; Chan et al., 2006). The pore formations by PIN-based

peptides appear by a carpet-like model, following the intracellular mechanisms of

activity (Alfred et al., 2013b). Recently it has also been suggested that PIN-based

peptides may inhibit DNA synthesis (Haney et al., 2013). It is thus clear that further

work is required to determine the mechanism(s) of activity of the PIN proteins in

membrane interactions, relevant to both their effects on texture and any in vivo defence

roles


- 29 -

1.5.4 Possible interactions between PINs

A considerable number of studies on comparative genetic and protein expression have

suggested some co-operation or interdependence between the two PIN proteins. The

presence of both PINs in their functional form at the surface of starch is necessary for

the soft phenotype (Hogg et al., 2004) and with a single protein present; the result is an

intermediate grain texture (Feiz et al., 2009c; Wanjugi et al., 2007). It is likely that

PINA has a primary role in binding to the starch granule surface and also has a co-

operative role in stabilising the binding of PINB, as well as mediating its binding to

starch granules (Amoroso et al., 2004; Capparelli et al., 2003; Gazza et al., 2005).

Furthermore there is a possible minor role for PINB in assisting the binding of PINA to

the starch granule surface (Bhave and Morris, 2008b). Supportive roles for PINA and

PINB in binding to the starch granules have been hypothesised (Amoroso et al., 2004;

Swan et al., 2006b). Ziemann et al. (2008), using the yeast two-hybrid system, provided

evidence of physical interactions between PINA and PINB, suggesting that both

functional PINA and PINB are essential for soft texture and that interaction between

PINA and PINB may occur in the wheat kernel.

The requirement of both PIN proteins in polar lipid binding was shown in transgenic

wheat (Feiz et al., 2009c). PIN proteins were independent of each other in localising to

the surface of starch granules, but the greater binding to polar lipids was observed only

when both PINs were present (Feiz et al., 2009c). The occurrence of both wild type

PINs may explain the increase in the equilibrium surface pressure at air/water interphase

and the degree of penetration in lipid monolayers (Clifton et al., 2007a). Overall, the

different lipid binding behaviours of PIN proteins provide further insight into the impact

of hydrophobic and cationic amino acid residues on the functional properties of their

antimicrobial activity (Sanders et al., 2013). Based on these reports, the synergism

between wild type PINA and PINB seems important for grain hardness and possibly in

their antimicrobial activity.


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1.6 Plant expression system

Among various expression systems such as bacteria, yeast, mammalian cells and

insects, plants are becoming an attractive system for the production of recombinant

proteins for pharmaceuticals, industrial proteins, monoclonal antibodies and vaccine

antigens due to many advantages including safe usage, rapid scale-up, long-term storage

and lower production cost (Daniell et al., 2001, 2009b; Fischer and Emans, 2000;

Fischer et al., 2003, 2004, 2013; Horn et al., 2004; Howard and Hood, 2005; Klimyuk et

al., 2008; Ma et al., 2003, 2005; Rybicki, 2009; Schmidt, 2004 Thomas et al., 2011). In

addition, plant cells have the ability to perform post-translational modifications (PTM)

of proteins which are typical of eukaryotic organisms (Gomord and Faye, 2004; Mett et

al., 2008; Vitale and Pedrazzini 2005). PTM affects the proteins activities and functions

such as folding, stability, and solubility, and dynamic interactions with other molecules,

the major type of PTM for plant-made recombinant proteins being glycosylation

(Marino, 1991; Stulemeijer and Joosten, 2008; Webster and Thomas, 2012).

A wide variety of promoters have been used for protein expression in plants, including

constitutive, inducible and tissue-specific, native and synthetic promoters (reviewed in

Egelkrout et al., 2012). The most widely used promoter is the cauliflower mosaic virus

35S promoter (CaMV35S), which promotes high-level of gene expression through the

transcriptional terminator of the Agrobacterium nopaline synthase gene (nos)

(Barampuram and Zhang, 2011; Lee et al., 2008). Several molecular events such as

gene transcription, mRNA translation and protein accumulation affect overall success of

protein expression (Table 1.6).


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Table 1.6 Summary of factors affecting gene transcription, mRNA translation and protein accumulation Source: modified from Egelkrout et al. (2012) Factors affecting transcription rate Factors affecting translation rate Promoter selection Post-transcriptional gene silencing Transcription factor binding (TFB) sites Codon usage Upstream regulatory sequences (URS) mRNA stability

Intron-mediated sequences Recruitment and retention of translation-ralated protein

Transcriptional gene silencing (TGS) Factors affecting protein accumulation Pre-mRNA stability Protein stabilisation Transgene trait stacking Protein localisation The impact of the site of integration Protein modification The number of gene copies Cellular milieu

Successful recombinant protein accumulation in plant systems depends on several key

factors including suitable host tissue, sub-cellular location, the nature of the foreign

proteins, their possible effect on the host plant, as well as the post-translational

modifications and the degree of protein purification required, and based on these

factors, there are several options for protein expression in plant systems (Egelkrout et

al., 2012; Schiermeyer et al., 2004). These are discussed below.

1.6.1 Host plants

Protein expression platforms have been developed for a variety of host systems

including seed crops (corn, canola, soybeans, and rice) and leafy crops (tobacco, alfalfa,

lettuce), as well as plant cell cultures (tobacco, carrot, rice), hairy root cultures and

aquatic plants (Lemna minor), in addition to moss and green algae (Wilken and

Nikolov, 2012). One of the major plant systems that is used for the production of

recombinant proteins is tobacco, the advantage being high leaf biomass yields as it can

be cropped several times a year (Basaran and Rodriguez-Cerezo, 2008; Twyman et al.,

2003; Twyman, 2004; Wilken and Nikolov, 2012).

1.6.1.1 Nicotiana tabacum (Tobacco)

The genus Nicotiana belongs to the family Solanaceae, which includes 76 species

originating in North America, South America, Australia and Africa (Chase et al., 2003;

Knapp et al., 2004). Nicotiana tabacum is an amphidiploid/allotetraploid species with

48 chromosomes, generated from hybridisation between N. sylvestris and either N.

tomentosiformis Goodspeed or N. otophpra Grisebach, all of which originate from


- 32 -

South America (Kitamura et al., 2001; Lewis, 2011). Nicotiana tabacum as a model

plant in research provides many practical advantages for large-scale production of

recombinant proteins, including high biomass yield, low maintenance and high seed

production (Twyman, 2004). It is a non-food, non-feed crop, hence carries a reduced

risk of transgenic material contaminating food chains (Stoger et al., 2005; Twyman et

al., 2003; Twyman, 2004). Tobacco cultivars contain a high amount of nicotine and

other alkaloids; hence, it is necessary to remove these toxic components (Twyman,

2004). Ma et al. (1995, 1998) reported the first successful expression of the secretory

monoclonal antibody in transgenic N. tabacum. The two tobacco cell lines, BY-2

(bright yellow 2) and NT-1 (N. tabacum 1), are readily available for Agrobacterium-

mediated genetic transformation and have been optimised for rapid growth (Doran,

2013).

1.6.1.2 Nicotiana benthamiana

The non-cultivated tobacco species, Nicotiana benthamiana, is a unique Australian

native plant that may have resulted from hybridisation between N. suaveolens and N.

debneyi (Goodspeed, 1954; Goodin et al., 2008). It is an amphidiploid with 38

chromosomes (Goodspeed, 1954) and has been used as a host in the production of

monoclonal antibodies and vaccines. It is a suitable host species for producing

recombinant proteins using viral vectors such as the tobacco mosaic virus (TMV)

(Twyman, 2004; Yang et al., 2004). It can be transformed with high efficiency and

maintained easily, due to its short stature, short regeneration time and high seed

production, and is also useful for transient expression of genes (Goodin et al., 2008).

1.6.2 Transformation method and expression systems

Genetic transformation can be achieved by physical or biological methods. Physical

methods are usually used to transfer non-viral vectors by using a gene gun (biolistics)

and chemical polyethylene glycol (PEG) transformation or electroporation of

protoplasts (Barampuram and Zhang, 2011; Rao et al., 2009). The biological method of

genetic transformation of plant cells by Agrobacterium involves the transfer and

integration of a large tumor-inducing (Ti) or rhizogenic (Ri) plasmid resident in

Agrobacterium into the plant nuclear genome. Although several species of


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Agrobacterium are known, two species are predominantly used for transformation;

Agrobacterium tumefaciens (T-DNA transfer for generation of transgenic plants) and

Agrobacterium rhizogenes (Ri-plasmid transfers necessary DNA to generate transgenic

hairy roots) (Gelvin, 2003; Gleba et al., 2014; Rao et al., 2009).

The production of recombinant proteins in plants, also known as molecular farming, is

classified into two expression systems, i.e., stable or transient. Stable expression

involves integration of foreign DNA into the plant genome and transfer to the next

generations (Sharma et al., 2005). The storage organs such as seeds have been used for

the stable expression of proteins of interest (Scheller and Conrad, 2004). The

chloroplast stable transformation requires more than two months and nuclear stable

transformation that requires several months (Patiño-Rodríguez et al., 2013).

Transient expression systems include Agrobacterium-mediated and viral-based

transformation, wherein the foreign gene is not integrated in the genome is expressed

only for a few days after being introduced into the plant cell/tissue. The strategy relies

on A. tumefaciens, which contains transgenes; the bacteria are infiltrated directly into

the organs of interest by injection or by using a vacuum chamber (agroinfiltration)

(Klimyuk et al., 2014; Leckie and Stewart, 2011; Leuzinger et al., 2013;

Vaghchhipawala et al., 2011). Transient expression is generally used to verify the

activity of the expression constructs and generate small amounts of recombinant protein

for functional analyses (Twyman et al., 2003). Many results have been obtained so far

with viral backbones such as those of the RNA viruses, Tobacco mosaic virus (TMV),

Potato virus X (PVX) and Cowpea mosaic virus (CPMV) (Gleba et al., 2014;

Marillonnet et al., 2005). Transient expression using viral vectors is one of the best and

rapid methods for obtaining a high yield of protein expression (Egelkrout et al., 2012).

1.6.2.1 Transient expression using viral vectors

Transient, viral-based systems are amongst the best available systems and have the

advantage of being a rapid method for large-scale production of proteins such as

antibodies and vaccine antigens (Gleba et al., 2007; Pogue et al., 2002). The viral

vector system is based on two strategies: ‘full virus’ vectors used to express long


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polypeptides (at least 140 amino acids) and ‘deconstructed’ virus vectors which rely on

A. tumefaciens to deliver DNA copies of one or more viral RNA replicons to plant cells

(Gleba et al., 2007; Gleba and Giritch, 2012). One main system of deconstructed virus

vectors is the ‘magnICON®’ from ICON genetics, based on the tobacco mosaic virus

(TMV), but instead of supplying whole RNA or linear DNA, it is divided in different

modules captured between the left and right border of T-plasmid of A. tumefaciens

(Gleba et al., 2004; Gleba et al., 2014; Marillonnet et al., 2004). Three A. tumefaciens

cultures deliver different vector modules containing different viral components,

targeting and gene sequences and these pro vector 5’ and 3’ modules assemble within

the plant cell in the form of functional viral vector (Marillonnet et al., 2004). After

delivery by agroinfiltration, they assembled inside the cell with the help of a site-

specific recombinase to create an RNA replicon to produce the desired protein. The

advantage of this system is that the desired protein can be produced at high yield,

because of viral amplification (Marillonnet et al., 2004). Targeting the proteins into

different subcellular compartments can alter protein stability (Gils et al., 2005) e.g.,

chloroplasts can improve stability (Doran, 2006). The green florescence protein (GFP)

has been expressed in different sub cellular compartments of N. benthamiana using this

system, to produce recombinant protein at up to 80% total soluble protein (Marillonnet

et al., 2004; 2005). A considerable amount of research has been published on the use of

the magnICON® system (Gleba et al., 2004, 2005, 2007; Gils et al., 2005; Giritch et al.,

2006; Huang et al., 2006, 2010; Marillonnet et al., 2004, 2005; Santi et al., 2006, 2008;

Webster et al., 2009; Werner et al., 2011).

1.6.3 Subcellular location in a plant system for protein accumulation

The targeting of foreign proteins to the most appropriate subcellular compartment is

important for protein accumulation, stability, folding and posttranslational modification

(Twyman, 2004). The choice depends on the structural characteristics of the protein

and the use of appropriate signals (Benchabane et al., 2008; Faye et al., 2005). Several

subcellular compartments have been considered including the cytosol, endoplasmic

reticulum (ER), apoplast, vacuole and chloroplast (Benchabane et al., 2008; Ma et al.,

2003).


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1.6.3.1 Cytosol

Recombinant proteins are retained in the cytosol when no targeting signals are used.

Several studies have revealed recombinant proteins that remain stable within the cytosol

(De Jaeger et al., 1999; Michaud et al., 1998; Marusic et al., 2007). However, the

cytosol may not be the best location for several reasons: high levels of protease and

ubiquitin activity, the negative redox potential that may affect the correct folding of

proteins with disulphide bonds, and a lack of important co- and post-translational

modifications, such as glycosylation, which may affect folding, assembly and the

tertiary structural stability (Benchabane et al., 2008; Faye et al., 2005).

1.6.3.2 Endoplasmic Reticulum (ER)

Targeting proteins to the ER can lead to high yields of recombinant proteins in planta

(Conrad and Fiedler, 1998; Wandelt et al., 1992). Recombinant proteins entering the

endoplasmic reticulum (ER) can be retained here by adding (SEK/H/K)DEL retention

sequences to the C-terminal (Benchabane et al., 2008; Michaud et al., 1998; Mainieri et

al., 2004), in most cases a (K/H)DEL sequence (Benchabane et al., 2008; Ma et al.,

2003). The presence of molecular chaperones in the ER can have a role in disulphide

bond formation (Benchabane et al., 2008; Faye et al., 2005; Nuttall et al., 2002).

1.6.3.3 Apoplast

Proteins can be targeted into the apoplast via the secretory pathway (Benchabane et al.,

2008). The plant apoplast includes the cell wall matrix and intercellular spaces and has

a wide range of physiological functions (Witzel et al., 2011). The lack of KDEL in

endosperm-expressed protein resulted in targeting to the apoplast, where mostly the

recombinant protein accumulates in the space under the cell wall (Christou et al., 2004).

1.6.3.4 Vacuole

The vacuole is one of the alternative destinations for recombinant proteins produced in

plants (Benchabane et al., 2008). Additionally, it has several key roles in planta e.g.,

control of cell turgidity, levels of macromolecules, accumulation of toxic secondary

metabolites and storage of high-energy compounds in vegetative tissues (Benchabane et

al., 2008; Twyman, 2004).


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1.6.3.5 Chloroplast

The chloroplast is one of the cellular destinations in a plant expression system, through

the addition of an appropriate targeting signal peptide (Daniell and Dhingra, 2002;

Hyunjong et al., 2006). The recombinant proteins may be moved to the chloroplast.

Chloroplast transformation offers uniform expression rates, a high copy number of the

transformed genes (due to hundreds of plastids in a cell), co-expression of multiple

genes from the same construct, minimal transgene escape in the environment due to the

maternal inheritance of chloroplast DNA and minimal gene silencing (Daniell et al.,

2004). Post-translational modifications such as multimerisation and disulphide bridge

formation can occur in the stroma (Daniell, 2006; Parachin et al., 2012). The

chloroplast can thus be a suitable compartment without needing the cell secretory

pathway or modifications such as glycosylation; however, some endogenous proteases

can affect the stability and accumulation of recombinant proteins (Adam and Clarke,

2002). However, this may not be relevant issue for proteins expressed at very high

yield (Daniell, 2006).

In conclusion, plants can act as ‘biofactories’ for the production of recombinant

proteins, for reducing the production costs due to the low maintenance cost and high

quality of the therapeutic protein (Davies, 2010; Gomord and Faye, 2004; Gleba, 2007;

Ling et al., 2010; Mett et al., 2008; Melnik and Stoger, 2013). The use of plant systems

for production of AMPs is summarised as follows.

1.6.4 Plant systems for heterologous production of antimicrobial peptide

Antimicrobial peptides/proteins (AMPs) are unique biologically active molecules that

comprise defence systems against numerous pathogens such as bacteria, fungi, parasites

and/or viruses. Heterologous expression systems, including bacteria and fungi as host

cells, have been used for production of AMPs with different sizes, folds and

complexities (reviewed in Parachin et al., 2012). Plant systems have also been used as a

suitable source for the production of AMPs and can be directly used for crop

improvement without purifying the peptide or proteins (Desai et al., 2010; Giddings et

al., 2000). The heterologous production of radish Rs-AFP2 defensin in tobacco

protected the transgenic tobacco from Alternaria longipes (Terras et al., 1995), whereas


- 37 -

the alfalfa anti-fungal peptide (alfAPF) defensin reduced the infection in transgenic

potato plants against the fungal pathogen Verticillium dahliae (Gao et al., 2000).

Several AMPs cannot be produced in microbial systems due to their antimicrobial

activity and complex structures. For example, retrocyclin-101 (RC101) and Protegrin-1

(PG1) can be used as therapeutic agents against bacterial and/or viral infections; both

have not yet been produced in microbial systems, but have been expressed in the

tobacco chloroplast with a yield of around 20% of total soluble protein, and retention of

antiviral activity (Lee et al., 2011b). The large scale production of AMPs can be

applied in numerous industries such as biotechnology, pharmaceuticals, cosmetics and

food. However, the limiting factor is the high production costs of some AMPs due to

their molecular mass and protein folding characteristics. The AMP can aggregate or be

degraded by endopeptidases, which also limits their bioactivity. Choosing the right

system is thus essential for large scale production and functional recombinant AMPs.

Plant-based expression systems are a promising platform for further engineering, and

production of AMPs (Parachin et al., 2012). All AMPs heterologously produced in N.

tabacum are listed in Table 1.7.


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Table 1.7 AMPs heterologous produced in Nicotiana tabacum AMP transformed Source Objective Resistance against References

protegrin-1 (PG-1)

Synthesized peptide

Production peptide and antimicrobial activity

Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Mycobacterium bovis, Candida albicans

Patiño-Rodríguez et al. (2013)

Dermaseptin Lysozyme C4V3

Phyllomedusa sauvagii, Gallus gallus, Synthetic

Production and isolation of the peptide and vaccine

Anti-HIV activity, induction of mammalian immune response

Rubio-Infante et al. (2012)

EtMIC2 Eimeria tenella Peptide production

Eimeria tenella (chicken coccidiosis)

Sathish et al. (2011)

Retrocyclin-101 and Protegrin-1

Artificial AMP based on rhesus monkey (Macaca mulatta) circular minidefensins

Chloroplast genome transformation aiming peptide production

Erwinia carotovora, tobacco mosaic virus

Lee et al. (2011b)

NmDEF02 Nicotiana megalosiphon

Proof of concept for crop improvement

Phytophthora parasitica var. nicotianae, Peronospora hyoscyami f.sp. tabacina, Alternaria solani, Phytophthora infestans

Portieles et al. (2010)

Trichokonins Trichoderma pseudokoningii

Proof of concept for crop improvement

Tobacco mosaic virus Luo et al. (2010)

Pal and Cpl-1 Phages infecting S.pneumoniae

Chloroplast genome transformation aiming peptide production

Streptococcus pneumoniae Oey et al. (2009)

MsrA2 (N-methionine-dermaseptin B1)

Phyllomedusa sauvagei and Phyllomedusa bicolor

Gene stacking for crop improvement

Fusarium solani, Fusarium oxysporum, Alternaria alternata, Botrytis cinerea, Sclerotinia sclerotiorum, Pythium aphanidermatum, Pectobacterium carotovorum

Yevtushenko and Misra, (2007)


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1.7 Summary of the literature and aims of the research

1.7.1 Summary of the above literature

This chapter provided background information that addressed the basics of puroindoline

proteins (PINs) in wheat, one of the most widely used crops worldwide. The

relationship between Pin genes and grain texture was reviewed. The review also

addressed the antimicrobial activity and lipid binding properties of PIN proteins and

PIN-based peptides. However, the in vivo roles for both PIN proteins in wheat seed and

the effect of these hardness mutations in puroindolines on the grain hardness as well as

their antimicrobial activity remain unclear. Several studies have reported the co-

operative or interdependent interaction binding to the surface of starch granules to

confer the soft phenotype, the basis for which is also unclear.

PIN proteins aggregate in the presence of membrane lipids and in the absence of lipids,

these proteins can still aggregate, but no precise information is available on the

relationship between aggregation and interaction with lipids in the absence of three-

dimensional structure of the proteins. The high resolution structure of PINs are yet to

be solved, due to problems with their crystallisation and difficulties in obtaining a stable

non-aggregated solution required for NMR structural characterisation. It is important to

investigate the functionality of the PIN proteins in order to better understanding the

biological functions of this family of proteins for their use in biotechnology and

therapeutic applications. Plant molecular farming approach can be used for expressing

large quantities of proteins or peptide. The characterisation of the two PIN proteins in

expressed in such systems will be invaluable for addressing many of the above stated

issues.


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1.7.2 The aims of the project

The specific aims of this project are as follows:

1. To clone the Pin genes encoding the putative mature PINA and PINB in to the

magnICON® viral vector system and utilise A. tumefaciens infiltrations, to express and

optimize their transient expression for high yield in the leaves of Nicotiana

benthamiana.

2. To test the antibacterial and antifungal activities of recombinant PIN proteins purified

with affinity purification using His-tag and hydrophobic interactions systems.

3. To analyse the protein-protein interactions among the PIN proteins in planta using

the BiFC system based on pNBV vectors and co-expression of the silencing suppressor

protein p19.

4. To test the significance of the tryptophan-rich domain (TRD) of PINA and the

hydrophobic domain (HD) of both PINs in any interactions between the PINs using the

BiFC system.

CHAPTER 2

Materials and methods

Chapter 2 Materials and methods

- 42 -

Equipment and materials

2.1 Equipment

The instruments and major apparatus used in this work are listed in Table 2.1.

Table 2.1 Instruments and apparatus used

Equipment Purpose Manufacturer

MyCyclerTM Conducting the Polymerase Chain Reactions (PCR)

Bio-Rad, California, USA

PowerPacTM HC power supply Nucleic acid and protein gel electrophoresis

Mini-protean® Tetra cell Polyacrylamide gel electrophoresis (PAGE or SDS-PAGE)

Westen blotting Mini-Protein II Trans blot module

Transfer of proteins from gels to membrane

SmartSpec 3000 Spectrophotometer

Determining bacterial concentration (Optical Density or OD)

Gene pluser II Electroporation system for transforming various cell types

NanoDropTM 1000 Spectrophotometer

Quantification of nucleic acid concentration

Thermo Fisher, USA

Finnpipette (0.5-10,5-50,20-200 and 100-1000µL) Dispensing liquid

Microplate reader Absorbance detection for ELISA assays and microbial growth turbidity

Microscope (Axioskop 2 MOT Plus)

Visualizing the bimolecular fluorescence complementation (BiFC) assay results Carl Zeiss,

Germany Microscope camera (AxioCam HR) Imaging the BiFC assay results

SNAP i.d Blocking, washing and antibody incubations for western blot Merck Millipore,

Australia Amicon® ultra 0.5 centrifugal filter (3K device - 3,000 NMWL)

Concentrating dilute protein samples

Spectroline Ultra Violet (UV) transilluminators

Visualisation of green or yellow fluorescent protein (GFP or YFP) expression

Spectroline, USA

Chemidoc system Visualisation of gel images UVP,California, USA

End-over-end mixer Mixing of samples for extractions and purification Ratek, Australia

Plant growth cabinet Growth of wheat plants under controlled conditions

Thermoline, Victoria, Australia

Film processor CP1000 Developer for illumination of antibody-HRP labeled western blot Agfa, Australia

Kodak, BioMax light film For western blotting analysis Sigma, USA


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2.2 Commercial kits

The commercial kits and reagents used in study are listed in Table 2.2.

Table 2.2 Commercial kits

Kit/material Purpose Manufacturer Biomix™ Red (2×) PCR

Bioline, Australia HyperladderTMI (10000bp-200bp)

Molecular weight standard for agarose gel electrophoresis of DNA samples

Precision Plus Protein Dual Xtra Standards (250 kDa-2 kDa)

Molecular weight sizing on SDS-PAGE gels

Bio-Rad, California, USA

Butyl-Toyopearl Hydrophobic interaction protein purification

Tosoh Corporation, Japan

HiYield™ Gel/PCR Fragments Extraction Kit

Recover or concentrate DNA fragments from agarose gels, PCR or other enzymatic reactions

Real Biotech Corporation (RBC), Taiwan HiYield™ Plasmid Mini Kit Plasmid DNA isolation

HisPur™ Ni-NTA Resin Affinity protein purification Thermo Fisher, USA KOD Hot Start DNA polymerase PCR Novagen, Germany

PlatinumR Pfx DNA polymerase PCR Life Technologies,

Invitrogen, USA SYBR® Safe DNA gel stain Staining of DNA in agarose gels

RNase A RNA digestion during genomic DNA extraction Sigma, USA

Restriction endonucleases DNA digestion NEB, (Genesearch), Australia

Sheep blood Haemolysis assay Amyl Media, Australia

Calf intestinal alkaline phosphatase (CIAP) Dephosphorylation of DNA ends

Promega, Australia Wizard® Genomic DNA Purification Kit Genomic DNA extraction


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2.3 Primary and secondary antibodies

Table 2.3 Antibodies for western blot analysis

Antibody name Purpose Specific Manufacturer and

Catalog number

Durotest® S* To detect friabilin proteins

Monoclonal antibody Host: mouse

R-Biopharm Rhone Ltd., Glasgow, UK; P10

Anti-His To detect histidine tag containing proteins

Monoclonal antibody Host: mouse

Life Technologies, Invitrogen, USA; 37-2900

Anti-GFP To detect GFP protein

Monoclonal antibody Host: rabbit

Life Technologies, Invitrogen, USA; G10362

Anti-mouse Secondary antibody Polyclonal antibody Host: goat Sigma, USA; A0168

Anti-Rabbit Secondary antibody Polyclonal antibody Host: goat Sigma, USA; A0545

*The Durotest® S has a monoclonal antibody raised to detect ‘friabilin’ protein mixture from bread wheat. The kit is detailed later under western blotting section.

2.4 Commonly used buffers, other solutions and media

The common buffers, solutions and media were prepared according to the instructions

in Sambrook and Russell (2001). These are listed in Table 2.4. All aqueous buffers and

solutions were prepared with MilliQ water (Millipore, Australia) that also autoclaved.

Solutions were sterilised by autoclaving (121°C for 20 minutes), or filter sterilizing

through a 0.22 µm syringe filter. All glassware and appropriate disposable plastic ware

(such as PCR tubes, centrifuge tubes, tips) were also autoclaved before use.

Table 2.4 Composition of general buffers and solutions

Buffer and Solution Composition

Coomassie Blue staining solution 40% methanol, 10% acetic acid, 0.05% Coomassie Blue R-250

Coating buffer 15 mM Na2CO3, 35 mM NaHCO3, (pH 9.6) De-stain solution 50% methanol, 10% acetic acid Infiltration buffer for magnICON® system

10 mM 2-(N-Morpholino)ethanesulfonic acid (MES), 10 mM MgSO4 (pH 5.5)

Infiltration buffer for BiFC system 10 mM MgCl2 and acetosyringone to a final concentration of 200 µM

Isopropyl-β-D-thiogalactopyranoside (IPTG) 0.1 M in sterile MilliQ water

Phosphate-buffered saline (PBS) 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 (pH 7.4)

PBS containing Tween-20 (PBST) PBS with 0.1% (v/v) Tween 20 Milk-PBS-Tween 20 (MPBST) 0.5% milk powder, PBS, 0.05% Tween 20


- 45 -

Table 2.4 Composition of general buffers and solutions (continued)

Buffer and Solution Composition

Native-PAGE loading dye, 2x 125 mM Tris (pH 6.8), 50% glycerol, 0.02% bromophenol blue

SDS-PAGE loading dye, 5x 50 mM Tris-HCl (pH 8.8), 10% sodium dodecyl sulfate (SDS), 30% glycerol, 500 mM ß-mercaptoethanol, 0.02% bromophenol blue

TB buffer 10 mM Hepes, 15 mM CaCl2, 250 mM KCl, (pH 6.7) then add MnCl2 to a final concentration of 55 mM

TE buffer 10 mM Tris, 1 mM EDTA, (pH 8) Tris acetate EDTA (TAE) buffer, 50x 2 M Tris base, 6.5 M EDTA disodium salt (pH 8)

Tris glycine buffer, 10x 25 mM Tris, 192 mM glycine buffer (pH 8.8)

Transfer buffer, 1x 25 mM Tris, 192 mM glycine buffer, 20% (v/v) methanol

Running buffer, 1x 25 mM Tris, 192 mM glycine buffer, 0.1% SDS 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) 5% (w/v) in dimethylformamide (DMF)

Composition of buffers and solutions for sequencing BDT reaction buffer, 5x 400 mM Tris (pH 9), 10 mM MgCl

2

Sequencing reactions clean-up solution 0.2 mM MgSO4 in 70% ethanol

Media Name Composition Luria Bertani (LB) broth 10 g/L tryptone, 5 g/L yeast extract, 5 g NaCl Luria Bertani (LB) agar LB with 15 g/L agar added (for plates) Luria Bertani (LB) glycerol LB with 30% glycerol Mueller-Hinton Broth (MHB) 21 g/L Potato Dextrose Agar (PDA) 39 g/L

Super Optimal broth (SOB) 0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4

Super Optimal broth with Catabolite repression (SOC) SOB with 20 mM glucose (filter sterilized)

Antibiotic stock Carbenicillin 200 mg/mL in sterile MilliQ water Ampicillin

50 mg/mL in sterile MilliQ water Kanamycin Rifampicin Spectinomycin


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2.4.1 Protein purification buffers

Various buffers were needed to be prepared as per the protein purification protocol and

systems applied. These are listed in Table 2.5.

Table 2.5 Composition of protein purification buffers

His-tag purification buffer (Native condition) [Thermo Fisher, USA] Equilibration buffer 20 mM sodium phosphate, 300 mM sodium chloride with 10 mM imidazole, pH 7.4 Wash buffer 20 mM sodium phosphate, 300 mM sodium chloride with 25 mM imidazole, pH 7.4 Elution buffer 20 mM sodium phosphate, 300 mM sodium chloride with 250 mM imidazole, pH 7.4 His-tag purification buffer (denaturing conditions) [PropurTM, Denmark] Equilibration buffer 20 mM sodium phosphate, 300 mM sodium chloride , 6 M urea with 10 mM imidazole, pH 7.4 Wash buffer 20 mM sodium phosphate, 300 mM sodium chloride, 6 M urea with 25 mM imidazole, pH 7.4 Elution buffer 20 mM sodium phosphate, 300 mM sodium chloride, 6 M urea with 250 mM imidazole, pH 7.4 Hydrophobic Interaction buffer [Lee et al, 2011b] Extraction buffer Saturated ammonium sulphate, pH 7.8; a final saturation of 60%, 70%, 80% Equilibration buffer 20% ammonium sulphate in 10 mM Tris–HCl, pH 7.8 Elution buffer 10 mM Tris–HCl, pH 7.8

2.5 Microbial strains

2.5.1 Strains used as hosts for gene cloning

Escherichia coli Mach1 strain was obtained from stock in Monash University and used

for general molecular cloning procedures. It was typically used for gene cloning.

Agrobacterium tumefaciens strain GV3101 was also used in transformation of plant

expression system vectors which is resistant to rifampicin due to a chromosomal

mutation (Lee and Gelvin, 2008).

The gift glycerol stock vectors which are detailed below were cloned in E. coli XL1-

Blue and A. tumefaciens strain LBA4404.


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2.5.2 Strains used for testing of antimicrobial activity of recombinant PINs

The bacterial species Escherichia coli (ATCC 25922) and Staphylococcus aureus

(ATCC 25923) and the fungal species, Collectotrichum graminicola, Drechslera brizae,

Fusarium oxysporum, Rhizoctonia cerealis and Rhizoctonia solani were obtained from

the culture collection at Swinburne University of Technology. The bacterial cultures

were streaked on fresh LB plates and incubated overnight at 37°C and a single colony

was used to inoculate overnight cultures. The fungal cultures were maintained on PDA

plates (Table 2.4).

2.6 Plant material

2.6.1 Propagation of wheat seedlings

The soft common wheat (Triticum aestivum L cv. Rosella) was used for isolation of the

wild type alleles of Puroindoline a (Pina-D1a) and Puroindoline b (Pinb-D1a)

henceforth designated as Pina and Pinb. The seeds, kindly provided by the Australian

Winter Cereals Collection (AWCC, Tamworth, Australia) were soaked in sterile water

overnight then sown on a sterilised mixture of potting soil and vermiculite (2:1). The

seedlings were grown in a plant growth cabinet (Thermoline, Australia) with 16 hours

of light (25ºC) and 70% humidity (Figure 2.1). For DNA extractions, leaf tissue was

harvested from 4 week old plants for immediate use or snap-frozen in liquid nitrogen

and stored at -80ºC.

2.6.2 Propagation of Nicotiana benthamiana seedlings

Nicotiana benthamiana was used for plant transformations. The seeds for the wild type

N. benthamiana in Australia were kindly provided by Dr Diane Webster, Monash

University, Melbourne. The seeds were germinated in jiffy peat pellets. The plantlets

(Figure 2.1) were transferred to sterilised mixture of potting soil and vermiculite (2:1)

and grown in a greenhouse maintained at 25°C with a 16h light/8h dark photoperiod.

Young, fully expanded leaves of 8 week old seedlings were used for infiltration.


- 48 -

A B

C D Figure 2.1 Wheat and N. benthamiana seeds and plantlets. A: Wheat seeds (T. aestivum L cv. Rosella); B: Wheat plantlet (3 weeks); C: N. benthamiana seeds; D: N. benthamiana plantlet (3 weeks). 2.7 General molecular methods

2.7.1 Genomic DNA extraction

Genomic DNA (gDNA) was extracted from the leaves of the wheat (T. aestivum L cv.

Rosella) seedlings (grown as above) using the Wizard® Genomic DNA Purification Kit

(Promega, Australia), according to the protocol supplied. Approximately 100 mg of the

leaf tissue was snap-frozen in liquid nitrogen, ground to a powder using a mortar and

pestle, mixed with 600 µL Nuclei Lysis solution and incubated at 65°C for 15 minutes.

Then 3 μL of RNase A (4 mg/mL) was added and the solution incubated at 37°C for 10

minutes. 200 μL Protein Precipitation solution was added and the mixture centrifuged

at 10000×g for 5 minutes. The supernatant was carefully mixed with 600 µL

isopropanol and centrifuged as above for 5 minutes. The DNA pellet was washed in

400 μL of 70% ethanol and re-spun as above. The DNA pellet was air-dried, dissolved

in 70 μL TE buffer and stored at -20°C. Then 5 μL of the DNA solution was run on a

1% agarose gel in TAE buffer to determine quality, any degradation and approximate

size.


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2.7.2 Plasmid DNA purification

The HiYield™ Plasmid Mini Kit (RBC, Taiwan) and the related instructions were used

for all plasmid preparations. A single colony of a bacterial culture was inoculated into 3

mL LB with relevant antibiotics (carbenicillin) and grown overnight at 37°C

temperature and 220 rpm. The cultures were pelleted for one minute at 200×g. The cell

pellets were resuspended in 200 µL PD1 buffer (Resuspension solution), mixed with

200 µL PD2 buffer (Cell Lysis solution) and incubated at room temperature (RT) for

two minutes. Subsequently, 300 µL PD3 buffer (neutralisation) was added to the lysate

and the solution centrifuged for three minutes at 200×g. The supernatant was

transferred to a spin-column assembly (provided with the kit) and centrifuged at 200×g

for one minute. The flow-through was discarded and the column washed twice with

diluted W1 and Wash buffers. The plasmid DNA was eluted by addition of 30 µL

Elution buffer to the column and centrifugation at 200×g for two minutes. DNA (5 µL)

was electrophoresed to check it quality and the DNA concentrations determined using a

Spectrophotometer. The methodology detailed above was applied to all non-

recombinant or recombinant plasmid preparations from transformants in E. coli Mach1

or E. coli XL-1 Blue.

2.7.3 Spectrophotometric quantification of DNA

Purified genomic DNAs were diluted 1:50 with sterile MilliQ water to determine the

DNA concentrations based on absorbance recorded at 260 nm and 280 nm using the

SmartSpec 3000 Spectrophotometer (Bio-Rad, USA), which also reported the

concentration based on absorbance of 1 at A260=50 μg/mL of double stranded DNA.

The A260/A280 absorbance ratio of 1.8 to 2 was used as an indication of high purity

(Sambrook and Russell, 2001). Purified plasmid DNA concentrations were determined

with 1 µL of sample using NanoDropTM 1000 Spectrophotometer.

2.7.4 Agarose gel electrophoresis

Agarose gel electrophoresis was used for multiple purposes such as qualitative

examination of the isolated gDNA, its approximate quantification, determining the sizes

of PCR products or restriction fragments, and purifying selected bands. Agarose gels

were typically prepared as 1% (w/v) gels in 1×TAE buffer with SYBR® Safe DNA gel


- 50 -

stain (0.5 μg/mL; Invitrogen) added to the cooled gel solution. Electrophoresis was

typically performed at 100V for 30-90 minutes. The DNA samples were mixed with a

5×loading dye and loaded onto gels. A molecular weight marker such as the

HyperladderTM

І (10,000-200bp; Bioline) was used for fragment size estimation and for

approximate estimation of DNA concentrations by comparison of the intensity of the

test DNA bands with that of the marker fragments. The gels were viewed on a UV

transilluminator and photographed using a Chemidoc system (UVP, USA).

2.8 The polymerase chain reaction (PCR) for gene amplifications

2.8.1 Design and synthesis of primers

Primers for gene amplifications by the polymerase chain reaction (PCR) were designed

using Netprimer (http://www.premierbiosoft.com/netprimer; last accessed October

2013) with the following criteria: length 18-25 bp (without tag), a 3’ G/C clamp,

percentage of GC content kept to approximately 50%, minimisation of secondary

structures such as dimers, cross-dimers and hairpins and the difference between

annealing temperatures of the forward and reverse primers limited to ±5°C. All primers

were synthesised commercially by Invitrogen (Australia) and supplied as desalted

pellets. The pellets were re-suspended in sterile MilliQ water to a stock concentration

of 2 μg/µL and stored at -20°C. The working concentration in standard PCR was 0.1

μg/µL (approximately 10 µM depending on primer length and sequence). The specific

primers designed for amplifying the Pina and Pinb genes and various protein expression

strategies are detailed in separate sections below.

2.8.2 Design of primers for amplification of full-length Pin genes

Primers Pina-D1-Forward and Pina-D1-Reverse were designed to amplify the entire

coding sequence (start to stop codon) of the wild type (WT) allele Pina-D1a of the Pina

gene (Gautier et al., 1994) (Genbank accession number DQ363911) (Table 2.6).

Likewise, Pinb-D1-Forward and Pinb-D1-Reverse were designed to amplify the entire

coding sequence of the WT allele Pinb-D1a (Gautier et al., 1994) (Genbank accession

number DQ363913).


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Table 2.6 Primer used for the wild type full length Pina-D1 and Pinb-D1 amplifications Amplicons Primers sequence* Expected product size

Pina-D1 Pina-D1-Forward

5’-ATGAAGGCCCTCTTCCTCA-3’ 447bp (Genbank: DQ363911) Pina-D1-Reverse

5’-TCACCAGTAATAGCCAATAGTG-3’

Pinb-D1 Pinb-D1-Forward

5’-ATGAAGACCTTATTCCTCCTA-3’ 447bp (Genbank: DQ363913) Pinb-D1-Reverse

5’-TCACCAGTAATAGCCACTAGGGAA-3’ *Start and stop codons are shown in bold

2.8.3 Design of primers for directional cloning into magnICON® and BiFC vectors

These are described in chapter-specific methods sections below.

2.8.4 Typical PCR conditions

Routine amplifications such as those of Pina and Pinb genes from gDNA or inserts

from recombinant plasmids, were generally carried out in 25 µL volumes, consisting

12.5 μL of 2×BioMixTM Red (Bioline, Australia), 5-10 ng plasmid DNA or 100 ng of

first-round PCR products as templates, and 0.1 µg of each forward and reverse primer.

Negative control reactions were identical except that the template DNA was omitted.

All amplifications involved an initial denaturation at 94°C for 5 minutes. This was

followed by 35 cycles of denaturation at 94°C (30 seconds), annealing at primer-

specific temperature (30 seconds) generally 5°C below the theoretical annealing

temperatures for both primers, extension at 72°C (30 seconds) and then a final extension

at 72°C for 5 minutes. The extension time was estimated roughly using 2 kb/min as the

rate of Taq polymerase activity at 72°C. The BioMixTM Red contains an inert red dye

that permits easy visualisation and direct loading onto a gel. Five μL aliquots of the

PCR products were examined by gel electrophoresis.

2.8.5 PCR for directional cloning of Pin genes into plant expression vectors

The PCRs for amplifying the mature protein-encoding sections of Pin genes (i.e.,

without the putative signal peptides; Gautier et al., 1994) for directional cloning into

various expression vector systems (described later) were conducted using KOD Hot

Start DNA Polymerase (Novagen, Germany) for increased specificity. For each

reaction, the following was assembled in a 0.5 mL PCR tube: 5 µL 10×PCR buffer for

KOD Hot Start DNA polymerase, 0.2 mM dNTPs and 1 mM MgSO4, 200 ng gDNA or


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5-10 ng plasmid DNA, 0.5 U/μL of KOD Hot Start DNA Polymerase, each primer to a

final concentration of 0.3 μM and sterile MilliQ water to a final volume 50 µL.

Negative controls (no template) were also included. The polymerase was activated by

heating for 2 minutes at 95°C followed by 35 cycles of denaturation at 95°C (20

seconds), annealing at 60°C (10 seconds) and extension at 72°C (15 seconds). The

results were assessed by agarose gel electrophoresis using 5 µL of each PCR products.

2.8.6 Purification of PCR products

The PCR products (when of a single size) or DNA bands of interest were purified using

HiYield™ gel/PCR fragments extraction kit (RBC, Taiwan). For purification from gels,

the desired band was excised and weighed (maximum 300 mg in one tube). The gel

slice was mixed with 500 µL of DF buffer and incubated at 55°C for 10 minutes to melt

the gel. The mixture was transferred to a spin column assembly (provided with the kit)

and centrifuged at 6,000×g for 30 seconds. The column was then washed with 500 µL

wash buffer. The assembly was centrifuged for additional 30 seconds at 10,000×g to

ensure wash buffer was removed completely. The elution buffer (20 µL) was then added

to the centre of the spin column, incubated for 2 minutes at RT and centrifuged at

10,000×g for 2 minutes. The eluted purified DNA was stored at -20°C. The kit was

also used for direct clean-up of PCR products, prior to restriction digestion and/or

cloning. The reaction (up to 100 µL) was transferred to a microcentrifuge tube. Five

volumes of DF buffer were added and mixed, followed by the steps as above. The

eluted DNAs were quantified by gel electrophoresis and/or NanoDropTM 1000

spectrophotometer.

2.9 Cloning and DNA sequencing

2.9.1 Restriction enzyme digestions of PCR products and vectors

A restriction digest typically contained 100-200 ng DNA substrate, 1 μL of a restriction

enzyme (typically 20 U/μL; New England Biolabs), 10 μL of the appropriate NEB

buffer, 1 μL BSA (10 mg/mL; New England Biolabs) and sterile MilliQ water. For in

planta expression of putative mature PIN proteins, the PCR products of full-length Pina

and Pinb genes were re-amplified with primers containing added restriction sites (and

additional tags) for directional cloning into various expression vectors (detailed in


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sections 2.13.3 and 2.14.4). These second-round PCR products and the corresponding

vectors were double-digested with two appropriate enzymes together to enable their

ligations. All reactions were incubated at 37°C for 2 hours. An aliquot was tested on an

agarose gel to check the success of digestions.

2.9.2 Ligation reactions

Ligations were typically performed in 10 µL volumes containing 1 µL ligase buffer

(10x), 1 µL rapid T4-ligase (NEB), 50 ng digested plasmid DNA vector and 200 ng

insert (e.g., the purified or restriction-digested PCR products). For an efficient ligation,

the amount of plasmid vector should be one third of the insert, in terms of the number of

molecules. The amount of insert was calculated as per Sambrook and Russell (2001):

Insert (ng) = Vector (ng)×Insert (Kb)Vector (Kb)

×molar ratio InsertVector

.

Ligation reactions without insert were used as negative controls. The reactions were

held at 4°C overnight and then transformed into chemically competent E. coli Mach1

cells.

2.9.3 Preparation of chemically competent cells of E. coli Mach1

Chemically competent E. coli Mach1 cells were prepared according to Inoue et al.

(1990) with minor modifications. A single colony from a LB agar plate was inoculated

into 5 mL LB medium and incubated overnight at 37°C on a shaker (200 rpm). This

culture was used to inoculate 250 mL of SOB medium and incubated at 37°C with

shaking until the cell density reached OD600

= 0.5. The culture was cooled on ice for 10

minutes then 40 mL of it was transferred into 50 mL tubes, followed by centrifugation

at 2,500×g for 10 minutes at 4°C. The pellets were resuspended in 12 mL ice-cold TB

buffer and incubated on ice for 10 minutes. The washing step was repeated and the

cells were resuspended in 3 mL of TB buffer. This was followed by the addition of

DMSO to a final concentration of 7% (v/v) with gentle mixing and incubation on ice for

10 minutes. The cell suspension was dispensed into 0.1 mL aliquots into prechilled

tubes, which were then snap frozen in liquid nitrogen and stored at -80°C.


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2.9.4 Transformation of E. coli Mach1 competent cells by heat shock

A tube of the above cells was thawed on ice for 5 minutes and 10 µL of a ligation

reaction was added to it. The suspension was held on ice for 20 minutes, followed by

heat-shock at 42°C for 50 seconds, then holding on ice for 2 minutes. The cells were

transferred into 0.5 mL SOC medium and incubated at 37°C for 1 hour with shaking

(200 rpm) then spread onto LB agar containing 100 µg/mL of carbenicillin and

incubated for 16 hours at 37°C.

2.9.5 Screening of E. coli transformants containing recombinant plasmids by

colony PCR

The recombinant colonies containing inserts in pICH-11599 or pNBV vectors cannot be

distinguished from non-recombinant ones with blue/white selection, as the pICH vector

does not carry the lacZ gene for the α-peptide of ß-galactosidase and the lacZ gene is

also not active in pNBV. Hence colony PCR was used, wherein half each of many

individual (all white) colonies from E. coli transformants were picked from the LB

plates and transferred into 25 µL 2x Red PCR mix and the DNA amplified using gene

specific primers (Table 2.7 and 2.11). The results were assessed using gel

electrophoresis. For the colonies which were found positive for inserts, the remaining

half colony was inoculated into 5 mL LB broth with the appropriate antibiotic and

grown overnight at 37°C with shaking (200 rpm) and used for plasmid purification

(Section 2.7.2).

2.9.6 Preparation of electro-competent A. tumefaciens cells

Agrobacterium strain GV3101 cells were prepared as per Mattanovich et al. (1989) with

minor modifications. The stock culture was streaked onto a non-selective LB agar agar

plate and grown for 1.5 days at 28°C with shaking (200 rpm) in 10 mL LB media

containing 50 µg/mL rifampicin. One mL of this culture was used to inoculate 100 mL

LB medium containing 50 µg/mL rifampicin and incubated at 28°C with shaking until

the cell density reached OD600=0.6. The culture was chilled before centrifugation of 25

mL of it at 2000×g for 20 minutes at 4°C. The supernatant was discarded and the cell

pellet resuspended in 25 mL of ice cold 1 mM HEPES buffer (pH 7) and centrifuged.

The cells were resuspended gently into 2 mL ice-cold glycerol (10%) and then a further


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10 mL glycerol (10%). The cells were pelleted again and resuspended in 300 µL of

glycerol (10%) with gentle mixing then aliquoted into 45 µL batches in pre-chilled

tubes, which were stored at -80°C.

2.9.7 Electroporation of electro-competent A. tumefaciens cells

Electroporation was performed in 2 mm electroporation cuvettes using 1 µL of (non-

recombinant or recombinant) plasmid DNA and 45 µL electro-competent

Agrobacterium cells prepared as above. After mixing above, the cuvettes were

incubated on ice for 5 minutes, then put into the electro holder of Gene Pulser (Bio-Rad,

USA) and pulsed at 2.5kv (kilovolts), 200Ω (ohm), 25μF (Microfarad), 5.7msec

(millisecond). One mL LB was immediately added into the cuvette. Cells were

transferred into a culture tube and shaken at 28°C for 60 minutes. All non-recombinant

magnICON® modules (extracted earlier from their E. coli XL1 Blue hosts) and all

recombinant pICH11599 vectors were electroporated individually into A. tumefaciens,

plated on plates with 100 µg/mL carbenicillin plus 50 µg/mL rifampicin selections and

grown at 28°C for two days. Recombinant pMLBART vectors were also electroporated

and grown on 50 µg/mL spectinomycin plus 50 µg/mL rifampicin selections at 28°C for

two days.

2.9.8 Screening of A. tumefaciens transformants containing recombinant plasmids

by colony PCR

Colony PCR was used to identify colonies of A. tumefaciens containing inserts. Half

each of many individual colonies from A. tumefaciens transformants were picked from

the LB plates and transferred into 25 µL 2x Red PCR mix volume and the DNA

amplified using gene specific primers as above. The results were assessed using gel

electrophoresis. The remaining half-colonies of interest were inoculated into 5 mL LB

broth with the appropriate antibiotics and grown overnight at 28°C with shaking (200

rpm) for preparing the glycerol stock (750 µL of bacterial culture plus 750 µL LB

glycerol) and processing for infiltration to the plant.


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2.9.9 DNA sequencing

All plasmids from E. coli Mach1 were prepared using HiYield™ plasmid mini kit

(RBC, Taiwan), as mentioned earlier. These were sequenced using the BigDye

Terminator (BDT) version 3.1 (Applied Biosystems, USA) according to the instructions

of the Australian Genome Research Facility (AGRF, Melbourne, Australia). The

reactions contained 1 μL BDT reagent, 2 μL 5×BDT buffer, 200-500 ng plasmids DNA

template and 3.2 pmol sequencing primer (Pin gene based primer), made up to 10 µL

with sterile MilliQ water. The cycling conditions were as follows: initial denaturation at

96°C for 2 minutes then 35 cycles of denaturation at 96°C for 10 seconds, primer

annealing at 50°C for 5 seconds and extension at 60°C for 4 minutes. Following this,

the reactions were cleaned up by the addition of 75 μL of 0.2 mM MgSO4

ethanol

solution, incubated at RT for 15 minutes and centrifuged at 200×g for 20 minutes. The

pelleted DNA was washed with 100 μL 70% ethanol followed by centrifugation using

the same conditions. The pellets were air-dried and resuspended in 20 μL sterile MilliQ

water. The solution was submitted to the AGRF (Melbourne) for capillary separation

using an AB3730x/DNA Analyser. The resulting chromatograms were inspected

visually using BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) for quality

checking. Generally, sequencing was done in both forward and reverse directions.

2.10 Bioinformatics methods

2.10.1 Sequence alignments

The putative amino acid sequences were translated from the nucleotide sequence data

and aligned using Bioedit v 7.1.11 sequence alignment editor program. Bioedit also

was used for analysis of gene sequences of Pina-D1, Pinb-D1. The alignments of DNA

sequences were performed with homologous sequences found in Genbank

(http://www.ncbi.nlm.nih.gov/genbank).

2.10.2 Plasmid vector maps

Vector maps of pNBV and pMLBART plasmids were created using A plasmid Editor

(ApE) software v 8.5.2.0 (http://biologylabs.utah.edu/jorgensen/wayned/ape; last

accessed March 2014) for sequence annotation, virtual digests and circular schematic

sequence representation.


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2.10.3 Predictions of the isoelectric point (pI) and molecular weight (Mw) of PIN

proteins

The pI and Mw of the putative mature PINA and PINB sequences were determined

using the ‘compute pI/Mw tool’ at the Expert Protein Analysis System (ExPAsy)

(http://web.expasy.org/compute_pi/).

2.10.4 Prediction of protein subcellular localisation

The subcellular locations of mature PINA and PINB were predicted for plant systems

using WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html), which is an

extension of the PSORT II program for converting protein amino acid sequences into

numerical localisation features; based on sorting signals, amino acid composition and

functional motifs (Horton et al., 2007).

2.10.5 Modeling and structure prediction of proteins

The putative amino acid sequences of wild type PIN proteins were submitted to the

Iterative Threading Assembly Refinement (I-TASSER). Using this method, three

dimensional (3D) atomic models were generated from multiple threading alignments

and iterative structural assembly simulations (Roy et al., 2010; Zhang, 2008). The

structures were visualized using molecular visualisation system and the final structural

models with tryptophan-rich domain (TRD) and hydrophobic domain (HD) produced

using PyMOL (http://www.pymol.org/; last accessed January 2014) (Delano, 2002).

The output of the I-TASSER server included the following: five full length atomic

models (ranked based on cluster density), the images of the predicted models and their

estimated accuracy (including confidence score of all models and prediction of TM-

score and RMSD for the first model), the predicted secondary structures, predicted

solvent accessibility and 10 proteins in PDB (Protein Data Bank) which are structurally

closest to the putative models (Roy et al., 2010). The ‘C-score’ is a confidence score

for estimating the quality of the predicted models by I-TASSER and is calculated based

on the significance of the threading template alignments and the convergence

parameters of the structure assembly simulations and is highly correlated with the TM

(template modelling)-score and RMSD (root-mean-square-deviation). The latter two

are used as standards for measuring similarity between two structures, and are useful in


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determining the accuracy of structure modelling when the native structure is unknown.

A TM-score > 0.5 indicates a model of correct topology and TM-scores < 0.17 indicates

random similarity (Roy et al., 2010).

2.10.6 Protein-Protein complexes structure prediction

Threading-recombination approach, (http://zhanglab.ccmb.med.umich.edu/COTH/)

(CO-Threader: COTH) (Mukherjee and Zhang, 2011) is based on to fold-recognition

and structural recombination of protein-protein complexes prediction by combining

tertiary structure templates with complex alignments. The sequences are first aligned to

complex templates using a modified dynamic programming algorithm. The monomer

templates are then structurally superposed to the dimer template to generate the final

models. The final protein-protein complex predictions for PINA+PINB and

PINA∆HD+PINB∆HD were produced using PyMOL.

2.11 Biochemical methods for protein analysis

2.11.1 Protein concentration measurements

Protein concentrations were measured according to Bradford (1976) using the Bradford

reagent (Bio-Rad, USA) for microtitre plate applications as described by the supplier.

The Bio-Rad dye reagent concentrate was diluted 1:5 with sterile MilliQ water and

filtered with Whatman filter paper 1. Bovine serum albumin (BSA) was used to

generate the standard curve using the range of 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL. Protein

samples (total soluble proteins or purified recombinant PIN proteins expressed in N.

benthamiana; described later) were diluted 1:10 and/or 1:50 in PBS. To each well in a

microtiter plate, 10 µL of each of the samples and standards (duplicates of each

dilution) was added, followed by 200 µL of diluted dye regent, and incubated for 10

minutes. The absorbance was measured using a Microplate reader (Thermo Fisher,

USA) at 595 nm wave length. The protein concentration of each unknown sample was

calculated from the standard curve.


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2.11.2 SDS-PAGE

Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was used

to assess protein expression profiles, sizes of the (denatured) expressed PIN proteins

and purity at various stages of purification. Samples were mixed with 5 µL of 5x

protein loading dye and boiled for two minutes. The proteins of interest were separated

using a 15% ReadyGel® Tris-HCl gel (Bio-Rad, USA) in running buffer at 20 mA for 1

to 2 hours. The gel was immersed in Coomassie Blue staining solution with gentle

rocking for one hour then immersed overnight in de-staining solution. A molecular

weight marker (250 kDa to 2 kDa) was used as size standards (Table 2.2).

2.11.3 Western blotting analysis for PIN proteins detection

Western blot analyses were carried out using the Durotest®S kit (R-Biopharm, Rhone

Ltd., http://www.r-biopharm.com/wp-content/uploads/4173/DUROTEST-S-Follow-On-

P10.pdf; last accessed February 2014). The kit has a monoclonal antibody raised

against friabilin and it is highly specific for the PIN proteins, which are the two major

components of friabilin (Greenwell et al., 1992; Rakszegi et al., 2009; Wiley et al.,

2007). The kit is commonly used in the pasta industry for rapid detection of

adulteration of durum wheat (semolina, which lacks PINs) with common wheat flour,

which contains the friabilin (mostly PINs). The kit contains two standards for the test:

(i) 100% durum flour, and (ii) durum flour containing 3% non-durum flour (i.e.,

common wheat flour). The epitope recognised by the Durotest antibody has not been

mapped (Greenwell et al. 1992; Wiley et al., 2007).

The total soluble proteins or purified recombinant PINs expressed in N. benthamiana

(described later) were identified through western blotting as described by Rakszegi et al.

(2009) and Wiley et al. (2007) with modification. Proteins was fractionated by SDS-

PAGE then transferred to polyvinylidene difluoride (PVDF) transfer membrane

(Millipore, Australia) in transfer buffer. The membrane was blocked in 0.5% MPBST

for one hour at 4°C on shaker. The Durotest antibody was used at a dilution of 1 in

1000 in 3 mL 0.3% MPBST and incubated for ten minutes at RT by using SNAP i.d.®

protein detection system (Millipore, Australia). The membrane was washed three times

with PBST, then incubated with the secondary antibody (the anti-mouse horseradish


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peroxidise (HRP) conjugate produced in goat) at a dilution of 1 in 1000 in 3 mL 0.3%

MPBST for ten minutes at RT by using SNAP id.® system. Following three washes

with PBST, the membrane was incubated in chemiluminescent detection reagents (ECL)

(Millipore, Australia) for five minutes and exposed to a Kodak, BioMax light film

(Sigma, USA) for 30 seconds or 1 minute and developed by AGFA CP1000 processor.

2.11.4 Enzyme-linked immunosorbent assay (ELISA) for approximate quantitation

of recombinant PIN proteins

Indirect ELISA involves two binding processes: binding of a primary antibody to the

antigen of interest coated to the microtitre plate surface, and binding of a conjugated

secondary antibody to the primary antibody, followed by assay of activity of the thus-

activated enzyme linked to the secondary antibody (Figure 2.2). The ELISA protocol

for PIN proteins was based on Zhang et al. (2010a) with modifications. The

concentration of PIN proteins expressed in the total soluble protein (TSP) extracts from

infiltrated N. benthamiana leaves (described later) were quantified approximately using

extract of 3% non-durum wheat (Durotest kit; R-Biopharm Rhone Ltd) with different

dilutions for developing a standard curve. The TSP samples were diluted 1/50, 1/100

and 1/200 in coating buffer. Each well (of a 96-well plate) was coated with 100 μL of

the diluted protein samples and left overnight at 4°C. The plate was washed three times

at RT with a phosphate buffered saline solution containing Tween-20 (PBST), then

blocked with 300 µL PBS containing 1% (w/v) bovine serum albumin (PBS-BSA) and

incubated for two hours at RT. Following three washes with PBST, 100 μL of the

Durotest antibody (diluted 1:1000 in PBS-BSA) was added and the plate incubated

overnight at 4°C. The plate was washed three times at RT with PBST. Then 100 μL

solution containing goat anti-mouse horseradish peroxidase (HRP) conjugated antibody

(Sigma, USA) was added (dilution 1:1000 in PBS-BSA) to the plate. The plate was

incubated at RT for one hour and then wells washed five times with PBST. One

hundred μL TMB (3, 3′, 5, 5′-Tetramethylbenzidine) (Sigma, USA) was added and

plates incubated for 15 minutes at RT. The reactions were stopped by addition of 100

μL H2SO4 (0.18 M) and absorbance was measured at 450 nm using a microplate reader

(Thermo Fisher, USA). The coating buffer and infiltrated leaf without Pin construct

were used as negative controls.


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Figure 2.2 Diagram of indirect ELISA assay. The antigen (Ag) of interest is coated directly to the assay plate and detection of the antigen performed using enzyme-conjugated (E) to the unlabeled primary and conjugated secondary antibodies.

2.12 Testing of antimicrobial properties of the in planta expressed PINs and a PIN-

based synthetic peptide

2.12.1 Design of synthetic peptide based on PINA

The peptide PuroA (FPVTWRWWKWWKG-NH2) was modeled on the TRD of PINA

and used as control for antimicrobial activity, as it had been tested previously (Jing et

al., 2003; Phillips et al., 2011). The peptide was synthesised at >95% purity by

Biomatik Corporation (Ontario, Canada) by solid-phase method using N-(9-flurenyl)

methoxycarbonyl (Fmoc) chemistry, with C-terminal amidation (NH2). Stock solution

was prepared at 1 mg/mL in 0.01% glacial acetic and stored at -20°C.

2.12.2 Antibacterial activity assay

Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used

to test the antibacterial activity of the PIN proteins expressed in N. benthamiana. The

minimum inhibitory concentration (MIC) was determined by the microtitre broth

dilution method (Wiegand et al., 2008) with modifications. Bacterial suspension from

an overnight culture was adjusted in Mueller-Hinton Broth (MHB; Oxoid, USA) to

1×108 CFU/mL

using a spectrophotometer (OD600= 0.08-0.13). The suspension was

diluted in MHB to 5×105

CFU/mL. Recombinant purified PIN, from the elution steps of

the His-tag purification and hydrophobic interaction systems (see later) in

approximately 1 mg/mL stock concentrations (measured by Bradford assays) were

added (25 μL) to the wells of sterile flat-bottomed 96-well plates (Corning, Australia)

Substrate

Ag

E

Primary antibody

Secondary antibody conjugate


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and a two-fold serial dilution was carried out across each row with a starting

concentration of 250 μg/mL and final concentration of 0.5 μg/mL. The wells were then

inoculated with 75 μL of the bacterial suspension and incubated overnight at 37°C. The

MIC values were determined by visual observation of bacterial growth as well as

measuring absorbance at 595 nm using a microplate reader (Thermo Fisher, USA). All

protein samples were tested in triplicate. The MIC was defined as ‘the lowest

concentration of antimicrobial agent that prevents visible growth of microorganism

under defined conditions’ (Wiegand et al., 2008).

2.12.3 Antifungal activity assay

The antifungal activity of PIN proteins was determined by microbroth assay (Broekaert

et al., 1990) with modifications. Filamentous fungal cultures of Colletotrichum

graminicola, Drechslera brizae, Fusarium oxysporum, Rhizoctonia cerealis and

Rhizoctonia solani were grown in Potato Dextrose Broth (PDB; Difco-BD, USA) for

seven days at 25°C with shaking (200 rpm). Mycelia were vortexed vigorously and the

resulting suspension adjusted with PDB to a concentration of 0.8-5×106 mycelia

fragments/mL (OD600 = 0.15-0.17). The suspension was diluted 1:50 with PDB to a

final concentration 0.4-5×104 mycelia fragments/mL (Espinel-Ingroff and Cantón,

2007). Sterile water (~200 μL) was added to the outermost wells of plates when testing

filamentous fungi, so as to reduce the effects of evaporation from the test sample wells

during the longer incubation times. Recombinant purified PINs, from the elution steps

of His-tag purification and hydrophobic interaction systems in approximately 1 mg/mL

stock concentrations (measured by Bradford assays) in 25 μL volumes were added to

the first empty well and a two-fold dilution was carried out across each row with a

starting protein concentration of 500 μg/mL and a final of 2 μg/mL. Test wells were

subsequently inoculated with 75 μL of the fungal mycelia and incubated at 25°C in the

dark for 5-7 days. Then, MIC values were measured which is defined as the lowest

value to completely inhibit fungal hyphal growth (Espinel-Ingroff and Cantón, 2007).

All protein samples were tested in triplicate.


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2.12.4 Stability testing of recombinant PIN proteins

Aliquots of the protein solutions (the same as those tested above) were stored at 37°C,

RT (between 20°C-25°C), 4°C, -20°C and -80°C for one week. Activity against E. coli

was then determined as above, each sample being tested in triplicate.

2.12.5 Haemolytic activity assay

The haemolytic activity of recombinant PINs, from the elution step of the His-tag

purification system in approximately 1 mg/mL (measured by Bradford assays) stock

concentrations was tested as described by Dathe et al. (1996) and Phillips et al. (2011).

The sheep red blood cells (RBCs) were isolated from defibrinated blood (Amyl Media,

Australia) by centrifugation of the whole blood at 1200×g at 4°C for 10 minutes. The

pellets were washed three times with PBS (pH 7.4) and resuspended in 2 mL PBS.

Then, 25 μL aliquots of the RBC suspension were mixed with different concentrations

of the purified PIN proteins and or the PuroA peptide (500-16 μg/mL) then incubated

for 1 hour at 37°C with gentle shaking. The samples were centrifuged at 3,000×g for 5

minutes and the absorbance of the supernatant measured at 540 nm. PBS-only (no

haemolysis expected) and 1% Triton X-100 (Sigma, USA) in PBS (100% haemolysis

expected) were included as negative control and positive control respectively.

Haemolytic concentration was defined as the concentration of the protein/peptide

required to haemolyse 50% of the RBCs (HD50).


- 64 -


2.13 Expression of PIN proteins in N. benthamiana using magnICON® viral vector

and characterisation of their biochemical and antimicrobial properties

2.13.1 Principles of the magnICON® viral vector systems for in planta expression

The magnICON® viral vectors system is based on tobacco mosaic virus (TMV) and

designed for in planta assembly from separate pro-vector modules to produce the

protein. The gene of interest is cloned in a 3’ vector module. Various 5’ modules are

then used for targeting the expressed protein to different parts of the plant cell (cytosol,

chloroplast or apoplast). An integrase module is used for recombination of 3’ and 5’

modules in planta. These modules recombine within the plant cell and the resulting

DNA is transcribed and the recombination sites (integrase) are spliced out, to create an

RNA replicon to produce the desired protein (Marillonnet et al., 2004) (Figure 2.3).

The protein is thus expressed in the appropriate subcellular compartment in the plant

cell (depending on the 5’ module used).

Figure 2.3 Schematic showing the assembly of the three viral vector modules and production of PIN proteins. The 5’ and 3’ modules with integrase in the plant cell allow recombining via the AttP and AttB sites. The intron is then spliced out. Viral replication occurs and translation and PIN protein processing occurs. Source: modified from Marillonnet et al. (2004).

+

5’ module P

3’ module PIN T

Integrase

5’ module P 3’ module PIN T

Recombination transcription

Splicing, export

Nucleus

5’ module 3’ module

Cytoplasm

Plant cell

Viral replication

Translation and protein processing

PIN

AttP AttB


- 65 -

AttB

intron

Splicing acceptor

site

KpnI NcoI EcoRI SacI BamHI HindIII

Nsil RB LB

The vectors were obtained from ICON Genetics (Halle, Germany;

http://www.icongenetics.com/html/home.htm; last accessed March 2014). The modules

are ampicillin or carbenicillin resistance in E. coli and come as follows (Figure 2.4 and

2.5).

(i) Two 3’ modules:

- [pICH11599]: 3’ module for cloning the gene of interest at the multi-cloning site

- [pICH7410]: Green Fluorescent Protein (GFP)-expressing 3’ module

Figure 2.4 The 3’ module pICH11599, for cloning in gene of interest at the multi-cloning site.

(ii) Various 5’ modules for targeting the expressed protein to different compartments of

the plant cell, including:

- [pICH15879]: untargeted (cytosol) 5’ module

- [pICH8420]: apoplast-targeting 5’ module

- [pICH12190]: chloroplast-targeting 5’ module

(iii) [pICH14011]: the integrase module for recombination of the 3’ and 5’ modules

within the plant cell

Tnos 3’ntr(cr) Tnos NPT Pnos

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB

AttB recombination site

NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP

Calreticulin apoplast targeting presequence

AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP

Artificial dicots chloroplast targeting presequence

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase

Nuclear localisation signal

Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP

RdRp (middle fragment)

RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F


- 66 -

Figure 2.5 The other magnICON® viral vector modules. Detailed vector maps for A: pICH15879, untargeted (cytosol) 5’ module; B: pICH8420, apoplast-targeting 5’ module; C: pICH12190, chloroplast-targeting 5’ module; D: pICH14011, the integrase module; E: pICH7410, GFP expressing 3’ module.

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F

pICH74107173 bp

Ap R

Kan

GFP

Intron

Trfa region

LB

RB

Pnos NTR

AttB

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (959)

BsaI (3859)

EcoRI (6983)

HindIII (1247)

Kpn I (6999)

PstI (1239)

Pvu II (6432)

XhoI (7010)

Nhe I (6785) Swa I (823)

Sca I (4278)

Nde I (1715)

pICH115996446 bp

Intron

Ap R

Trfa region

Kan

3'NTR

LB


NOS promoter

pUC ORI

RLK2 OriV

Nos term

Nos term

BamHI (7)

EcoRI (6434)

PstI (29)

PvuII (5741)

SacI (1)

ApaI (275)

HindIII (31)

NcoI (6426)

NheI (6094)

SalI (19)

ScaI (3587)

pICH842012679 bp

RdRp

AP r

Kan

MP

Intron (5' part)

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

AttP


AtAct2Prom

ORI

oriV

Nos term

Nos term

omega

BsaI (2907)

Kpn I (6047)

Nco I (11277)

Pvu II (5480)

XhoI (12482)

Not I (4352)

EcoRV (7528)

pICH1219012758 bp

RdRp

MP

Intron 5' part

Ap R

Trfa region

Kan

LB

Pnos

AttP


AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

BsaI (2894)

EcoRI (12273)

HindIII (9446)

Kpn I (6022)

PstI (10488)

Pvu II (5467)

Sac I (11242)

ApaI (1)

Sph I (7266)

Ava I (8443)

EcoRV (7503)

pICH140118816 bp

AP r

Kan

C31 integrase


Hsp81.1 promoter

RK2 TrfA

pUC ori

TDNA-LB

TDNA-RB

ORI

oriV

Nos term

Nos 3'

BamHI (1863)

ClaI (1155)

Kpn I (7925)

Nco I (1096)

PstI (12)

XbaI (6787)

XhoI (1)

Not I (6237) ApaI (2177)

Sph I (2342)

Nhe I (7718)

Sca I (5211)

FseI (1887)

Nde I (2648)

pICH1587913371 bp

MP


RdRP 5'

RdRP 3'

Intron 5'part

intron atbg3

intron atipl3

intron atsug1

intron atbg11

intron atatp2

Intron pet psk7-6-2

Ap R

Trfa region

Kan

LB

AttP

T-DNA RB

AtAct2Prom

pUC ORI

RLK2 OriV

Nos term

Nos term

omega

RdRp exon 10

RdRp exon 11

RdRp exon6

RdRp exon 7

BsaI (4219)

EcoRI (1022)

HindIII (11181)

Kpn I (7347)

Nco I (1)

PstI (12223)

Pvu II (6792)

SacI (13362)

ApaI (1326)

Sph I (8591)

Ava I (9768)

EcoRV (8828)

A B C

ED F


- 67 -

The above vector systems were used for the expression of the wheat Pina and Pinb

genes in N. benthamiana to study the localisation of the PIN proteins and purify and

characterise them. For this purpose, all above plasmids were extracted from their

glycerol stock cultures in E. coli XL1 Blue (provided by Dr Diane Webster, Monash

University) using the HiYield™ plasmid mini kit (Section 2.7.2). Four different 3’

expression modules; pICH-PINA-His, pICH-PINB-His, pICH-PINA-His-SEKDEL and

pICH-PINB-His-SEKDEL were then constructed in pICH-11599 such that they

contained the gene sections encoding the putative mature PINA or PINB protein, with a

C-terminal histidine tag (His-tag) or SEKDEL tag. The primer design and cloning

processes for this purpose are detailed below.

2.13.2 Design of primers for directional cloning into the 3’ module pICH11599

Primers were designed based on the open reading frame (ORF) encoding the mature

proteins, i.e., without the 28 and 29 residue signal peptides of PINA and PINB,

respectively (Gautier et al., 1994). For cloning of Pina, the nucleotide sequence

encoding the restriction site for EcoRI was incorporated at the 5’ end of the forward

primer and the sequence for BamHI incorporated at the 5’ end of the reverse primer.

For cloning of Pinb, the restriction site for NcoI was incorporated in the forward

primers and for SacI in the reverse primers. The reverse primer for both genes also

contained a sequence that would encode a C-terminal six-histidine tag (His-tag) in the

sense strand, to allow further protein purification. Further, another reverse primer was

designed for each gene such that it also encoded a further C-terminal SEKDEL (Ser-

Glu-Lys-Asp-Glu-Leu), the highly conserved eukaryotic endoplasmic reticulum (ER)

retention signal (Munro and Pelham, 1987; Wang et al., 2008). A BamHI site was

designed at the 5’ end of both the pICH-PINA-His-SEKDEL and pICH-PINB-His-

SEKDEL primers. A few extra spacer nucleotides were added to the primers before and

after the restriction site to allow proper digestions. The primers are listed in Table 2.7.

The expected PCR product sizes for both Pin genes with the His-tag and His-SEKDEL

tag are shown in Table 2.8.


- 68 -

Table 2.7 Primers used in directional cloning into 3’ viral module, pICH11599

Primer name Sequence of primers used* Cloning

sites

Pina-His Forward: 5’-GGTAGAATTCGATGGATGTTGCTGGCGGGGGTG-3’ Reverse: 5’-CCGGGATCCTCAATGGTGATGGTGATGGTGCCAGTAATAGCCAATAGTGCC-3’

EcoRI

BamHI

Pinb-His Forward: 5’-GAACCATGGAAGTTGGCGGAGGAGGTGG-3’ Reverse: 5’-CCGGAGCTCTCAATGGTGATGGTGATGGTGCCAGTAATAGCCACTAGG-3’

NcoI

SacI

Pina-His-

SEKDEL

Forward: 5’-GGTAGAATTCGATGGATGTTGCTGGCGGGGGTG-3’ Reverse: 5’CCGGGATCCTCATAGCTCATCTTTCTCAGAATGGTGATGGTGATGGTGCCAGTA3’

EcoRI

BamHI

Pinb-His-

SEKDEL

Forward: 5’-GAAGAATTCGATGGAAGTTGGCGGAGGAGGTGG-3’ Reverse: 5’CCGGGATCCTCATAGCTCATCTTTCTCAGAATGGTGATGGTGATGGTGCCAGTA3’

EcoRI

BamHI

* Cloning sites to be incorporated for directional cloning are shown in italics and shaded. Nucleotides in bold and shade are sequences encoding the His-tag in the sense strand. Nucleotides in bold and underline are sequences encoding SEKDEL in the sense strand. Nucleotides in italics and wave underline encode start and stop codons. The first residue of mature PINs is boxed.

Table 2.8 Genes cloned with His-tag and His-SEKDEL tag into pICH11599

Clones mature protein section of gene that was cloned into pICH11599 Final product size

pICH-PINA-His PINA ORF from DVAGGG to stop (363 bp) +His-tag (18 bp) 381 bp

pICH- PINA-His-SEKDEL

PINA ORF from DVAGGG to stop (363 bp) +His-tag (18 bp)+SEKDEL(18 bp) 399 bp

pICH-PINB-His PINB ORF from EVGGGG to stop (360 bp) +His-tag (18 bp) 378 bp

pICH-PINB-His-SEKDEL

PINB ORF from EVGGGG to stop (360 bp) +His-tag (18 bp)+SEKDEL (18 bp) 396 bp

2.13.3 Amplifications of Pin genes, restriction enzyme digestions, cloning into the

3’ module pICH11599, and sequencing of clones

The 447 bp full-length genes Pina-D1 and Pinb-D1 were amplified from the gDNA

of T. aestivum cv. Rosella (with primers given in Table 2.6).

These PCR products were then used as templates in second-round PCRs with the

primer combinations given in Table 2.7, to amplify the gene sections encoding the

mature PINs, with the His (and SEKDEL) tag(s) and the appropriate restriction sites.

Both second-round PCR products were purified using HiYield™ gel/PCR fragments

extraction kit (Section 2.8.6).


- 69 -

The purified PINA PCR products (PINA-His and PINA-His-SEKDEL) were each

double-digested with EcoRI and BamHI, as detailed in section (2.9.1).

The empty vector pICH11599 was purified from the stock culture in E. coli XL-1

Blue using HiYield™ Plasmid Mini Kit (Section 2.7.2).

An aliquot of pICH11599 was double-digested with EcoRI and BamHI.

The two double-digests of PINA were ligated separately with the EcoRI-BamH1

digested pICH11599.

Likewise, the purified PINB PCR product (PINB-His) and another aliquot of

pICH11599 were each digested with NcoI and SacI and then ligated with each other.

The purified PINB PCR product (PINB-His-SEKDEL) was double-digested with

EcoRI and BamHI and then ligated with the double-digested pICH11599.

The ligations were transformed into chemically competent cells of E. coli Mach1 (as

detailed in Section 2.9.4), and plated on LB agar supplemented with carbenicillin.

Several half-colonies per transformation were subjected to colony-PCR (Section

2.9.5) to identify those containing recombinant plasmids.

The other half-colonies of interet were cultured and plasmids were prepared from

these using HiYield™ plasmid mini kit (Section 2.7.2) and sequenced (Section

2.9.9)

The steps are summarized in Figure 2.6.


- 70 -

Figure 2.6 Generation of the Pin constructs in magnICON® viral vectors. A: the open reading frame of Pina and Pinb, encoding the mature proteins, followed by the His-tag sequence, ligated into; B: the magnICON® viral vector 3’ module; C: the open reading frame of Pina and Pinb, encoding the mature proteins, followed by the His-tag and SEKDEL sequence, ligated into magnICON® viral vector 3’ module. 2.13.4 Electroporation of recombinant pICH11599 and other modules into

A. tumefaciens

Following confirmation of correct DNA sequences encoding the mature PIN ORFs with

the C-terminal His or His-SEKDEL tags, the recombinant pICH11599 vectors were

electroporated into A. tumefaciens GV3101 (as described in section 2.9.7) and the

bacteria grown on LB medium containing 100 µg/mL carbenicillin plus 50 µg/mL

rifampicin selection at 28°C for two days. The colonies were screened by colony-PCR

for recombinant plasmids using specific primers (Table 2.7). All non-recombinant

vector modules were also electroporated individually into A. tumefaciens.


- 71 -

2.13.5 Agroinfiltration of N. benthamiana using the magnICON® viral vectors

Single colonies of A. tumefaciens cultures containing the different vectors were grown

in LB medium supplemented with carbenicillin (100 µg/mL) and rifampicin (50 µg⁄mL)

overnight at 28°C. Equal volumes (2.5 mL each) of three different cultures (Table 2.9)

(OD600=0.7 each) were mixed together in various combinations of (i) a 5’-module; (ii) a

3’-module; (iii) the integrase module. The mixture was then centrifuged at 600×g for

ten minutes. The bacterial pellet was resuspended in 10 mL of infiltration buffer (Table

2.4) and infiltrated into leaves of 8 week old N. benthamiana plants using a 1 mL

syringe without a needle (Figure 2.7). The injected area was marked with permanent

marker pen for ease of identification. Plants were allowed to grow for a further 8 to 10

days in green house conditions before the leaves were harvested and frozen in liquid

nitrogen then stored at -80°C.

Table 2.9 Tri-partatite infiltration of magnICON® constructs into N. benthamiana

Columns show specific infiltrated constructs. Rows show the Agrobacterium cultures containing different viral vector modules. The colors show the mixture of Agrobacterium cultures for specific infiltrated constructs. GFPc: GFP cytosol targeting; GFPch: GFP chloroplast targeting.

Agrobacterium Culture Infiltrated construct

Integrase Cytosol Chloroplast Apoplast GFP PINA

Or PINB

PINA-SEKDEL

Or PINB-

SEKDEL

Cytosol Chloroplast

Apoplast ER

GFPc GFPch

Negative


- 72 -

Figure 2.7 Agroinfiltration of N. benthamiana leaf. Lower surface of leaf was infiltrated with Agrobacterium suspension in infiltration buffer. 2.13.6 GFP expression

The green fluorescent protein (GFP) is 238 amino acid long and has MW of 26.9 kDa.

Shimomura et al. (1962) isolated GFP from the jelly fish Aequorea victoria and

demonstrated that it could exhibit bright green fluorescence when exposed to light in the

UltraViolet (UV) range. The gene for GFP was cloned by Prascher et al. (1992) and the

protein was expressed in E. coli by Chalfie et al. (1994). A 3’ viral vector containing

the GFP gene (pICH7410) was infiltrated into N. benthamiana leaves side by side with

the 5’ module for cytosol or the 5’ module for chloroplast. At 10 days post infiltration

(dpi), GFP expression in leaves was checked under UV light (Spectroline UV

transilluminator).

Protein methods

2.13.7 Extraction of total soluble protein (TSP) from N. benthamiana leaf

Pieces from the infiltrated area of the leaf (100 mg) were collected in pre-weighed two

mL tubes and the tubes weighed again. Stainless steel beads of 5 mm diameter (Qiagen,

USA) and 500 µL of extraction buffer [100 mM Ascorbic Acid, 0.1% Triton X-100,

EDTA-free protease inhibitor cocktail tablets in PBS, pH 7.4] were added to each tube.

Centrifugation at 600×g for ten minutes

Resuspended in 10 mL of infiltration buffer

Infiltrated behind the leaf of N. benthamiana using a 1 mL syringe

3’ module, 5’ module and integrase culture were mixed together

5’ module P 3’ module T

Att P Att B

Integrase


- 73 -

Leaf tissues were ground at 28 strokes per second for 1 minute using TissueLyserII

(Qiagen, USA), then centrifuged at 4°C at 10,000×g for 10 minutes. The extract was

filtered through Miracloth (Calbiochem, Australia) and centrifuged at 100×g for 5

minutes at 4°C. The clear supernatant was collected into a clean tube. The samples

were either stored at -80ºC or used directly for western blot analysis. In further

optimisations, different extraction buffers (Table 2.10) were used for TSP extraction

before western blot analyses.

Table 2.10 Protein extraction buffers used in this study

No Extraction buffers References

1 PBS containing 100 mM Ascorbic Acid, 0.1% Triton X-100 or Triton X-114, protease inhibitor cocktail tablet

Webster et al. (unpublished)

2 PBS containing 100 mM Ascorbic Acid, 20 mM EDTA, 0.1% Triton X-100, protease inhibitor cocktail tablet

Webster et al. (2009)

3 50 mM Tris (pH 8.8), 50 mM NaCl, 10 mM EDTA, 1 µg/mL leupeptin, protease inhibitor cocktail tablet

Sorrentino et al. (2009)

4 PBS containing 50 mM Tris (pH 8.8), 50 mM NaCL,

10 mM EDTA, 1 µg/mL leupeptin,100 mM ascorbic acid, 0.1% TritonX-100, protease inhibitor cocktail tablet

Tremblay et al. (2011)

2.13.8 Protein concentration measurements, SDS-PAGE, western blotting for

detection and ELISA for approximate quantitation of PINs

Protein concentrations of the TSP were measured by the Bradford assay (see section

2.11.1). SDS-PAGE was used to assess protein expression, the protein sizes and extent

of purification (Section 2.11.2). The PIN components in the TSP, or the purified

recombinant PINs from various methods described below, were identified through

western blotting (Section 2.11.3). The PINs expressed in the TSPs were quantified

approximately by ELISA (Section 2.11.4).

Methods for further PIN protein purification

2.13.9 His-tag purification under native conditions

The His-tagged recombinant PIN proteins expressed by the magnICON® viral vectors

were purified from the TSP using the immobilized metal affinity chromatography

(IMAC) system (Thermo Fisher, USA) which uses imidazole-containing buffers,

according to the manufacturer’s protocol with some modification. The Ni-NTA resin

was equilibrated with equilibration buffer (Table 2.5). TSP samples (20 mL) were


- 74 -

mixed with an equal volume of equilibration buffer, applied to the centrifuged tube with

pre-equilibrated resin and mixed on an end-over-end rotator for 30 minutes. The tube

was centrifuged at 700×g for 5 minutes at 4°C. The resin with the assumed bound His-

tagged protein was washed 3 times using two resin-bed volumes of wash buffer (Table

2.5) and centrifuged at 700×g for 2 minutes at 4°C. The bound proteins were eluted in

one resin-bed volume of elution buffer (Table 2.5) by centrifuging at 700×g for 2

minutes at 4°C. The eluted products were removed carefully and stored at -80°C.

Aliquots (50 µL) were taken at each step of purification for Bradford analysis, SDS-

PAGE and western blot analysis (as above).

2.13.10 His-tag purification under denaturing conditions

Urea [CO(NH2)2] is a powerful protein denaturant, as it disrupts the noncovalent bonds

in the proteins. Urea (6 M) was used for purification of the His-tagged recombinant

PINs, to solubilise and make the proteins more accessible for interactions with the Ni-

NTA resin. The TSP was purified using IMAC system (Nunc, Roskilde, Denmark)

under denaturing conditions with urea. The binding, wash and elution buffers with 6 M

urea and different concentration of imidazole were made up in PBS (pH 7.4) (Table

2.5). The TSP samples (20 mL) were applied to the pre-equilibrated nickel IMAC

column and centrifuged at 150×g for 30 minutes at 4°C. The column was washed 3

times using wash buffer and centrifuged at 500×g for 3 minutes at 4°C. Elution buffer

was added to the column and centrifuged at 500×g for 3 minutes at 4°C. The imidazole

and urea were removed from the final eluted product by dialysis (Section 2.13.12) and

stored at -80°C.

2.13.11 Hydrophobic interaction purification

The hydrophobic interaction purification method was used as described by Skosyrev et

al. (2003) and Lee et al. (2011) with modifications. Saturated ammonium sulphate (pH

7.8) was added to a final concentration of 60%, 70% or 80% to the TSP extract. This

suspension was then mixed vigorously with one-fourth the volume of ethanol (70%) for

2 minutes. The ethanol phase was separated by centrifugation at 2500×g at RT for 5

minutes and collected carefully to avoid disturbance of the interphase. Approximately

500 µL of 5 M sodium chloride (NaCl) solution was added to the ethanol phase and


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mixed for 2 minutes with one-fourth volume of n-butanol (Sigma, USA). The solution

was centrifuged at 2500×g for 2 minutes and the lower phase containing the fusion

protein was carefully collected. The lower phase solution was adjusted to 20%

ammonium sulphate and loaded directly on the pre-equilibrated column with Butyl-

Toyopearl (Tosoh Corporation; Japan). The Butyl-Toyopearl were equilibrated and

washed with equilibration buffer (Table 2.5). The protein was eluted with elution buffer

(Table 2.5) and stored at -80°C for SDS-PAGE and western blot analysis.

2.13.12 Dialysis

The eluted proteins under native and denaturing conditions were exchanged into PBS

(pH 7.5) to avoid precipitating the protein in imidazole and urea. The proteins purified

under denaturing conditions (in 6 M urea) were dialyzed against buffers containing

decreasing concentrations of the denaturing agent (urea) as follows: PBS, 6 M urea;

PBS, 3 M urea; and PBS, 1 M urea. Samples were dialyzed at 4°C for more than 8

hours in each dialysis buffer. Following dialysis, the Amicon ultra 0.5 centrifugal filter

device (3K-3,000 NMWL) were used to remove the rest of imidazole buffer by

centrifugation at 4,000×g for 15 minutes at 4°C for eluted proteins under denaturing

condition. The Amicon device was used for eluted proteins under native conditions to

remove imidazole buffer and change the buffer into PBS.

2.13.13 Concentrate of dilute protein samples

All diluted purified proteins were concentrated using Amicon ultra 0.5 centrifugal filter

device (3K-3,000 NMWL). Spin cycles were performed at 14,000×g for 500 μL of

sample. To recover the concentrated protein, the device was placed upside down in a

clean microcentrifuge tube and centrifuged at 1,000×g for 2 minutes at 4°C. The

samples were applied and the column centrifuged until sufficient concentration was

achieved.

2.13.14 Sodium phytate precipitation

Sodium phytate precipitation was performed as per Krishnan and Natarajan (2009), to

remove Rubisco from samples purified with the hydrophobic interaction system. PINs

purified with the hydrophobic interaction system were treated with 10 mM CaCl2 and


- 76 -

10 mM sodium phytate (phytic acid sodium salt hydrate) for 10 minutes at 37°C, and

then centrifuged at RT for 10 minutes at 10000×g. The supernatant was removed to a

fresh tube for further analysis.

2.13.15 Testing of antimicrobial and haemolytic properties of the expressed PINs

The antibacterial and antifungal properties of the recombinant PINs purified with His-

tag under native and denaturing conditions and the hydrophobic interaction purification

system were tested as detailed above (Section 2.12).


2.14 Protein-protein interactions of PIN proteins using BiFC system

2.14.1 Principle of the Bimolecular Fluorescence Complementation (BiFC) system

The BiFC system is used to identify interactions between two proteins. The technique

is based on the use of two candidate proteins that are translationally fused to the coding

region fragments of two non-fluorescent fragments of the yellow fluorescent protein

(YFP); YC (C-terminal half) and YN (N-terminal half). Therefore, in a BiFC assay,

interaction of the candidate proteins leads to functional YFP reconstitution and

fluorescence which can be detected by monitoring fluorescence via fluorescence

microscopy techniques (Hu and Kerppola, 2003; Kerppola, 2006) (Figure 2.8). Initially,

this technique was used to demonstrate protein-protein interactions in living mammalian

cells (Hu et al., 2002; Hu and Kerppola, 2003) and then adapted for the use in plant

systems (Bracha-Drori et al., 2004; Walter et al., 2004).


- 77 -

Figure 2.8 Principle of the BiFC assay. X and Y were proteins that fused to N-(YN) or C-(YC) terminal of the fluorescent protein (YFP) fragments. If X and Y are interacting proteins the fluorescent protein will be reconstructed and fluorescent signal detected. But if X and Y are non-interacting proteins, the N-(YN) and C-(YC) terminals will not be fused to reconstruct the fluorescent protein and no signal will be detected.

The BiFC test was applied here to test and visualize the occurrence of any interactions

between PINA and PINB proteins in living plant cells. The pNBV vectors used for this

purpose are described below.

2.14.2 New BiFC Vector (pNBV)

The pNBV (Figure 2.9) were kindly provided by Professor David Smyth and Mr Tezz

Quon (Monash University, Melbourne). The vectors (pNBV) contain the gene for

ampicillin or carbenicillin resistance, a terminator of the Nos gene (NosT) and 35S

promoter of the cauliflower mosaic virus. In each pNBV vector pair, the gene of

interest fused either to the N-terminal 155 amino acid fragment (YN) or to the C-

terminal 86 amino acid fragment (YC) of Yellow Fluorescent Protein (YFP) (Hu et al.,

2002).

YN

YC

Y

YN

YC

X X Y

YN

YC

X Y

YN

YC

Y X

Co-expression

Interaction

Co-expression

Interaction

Fluorescent signal

No signal


- 78 -

A B

C D

Figure 2.9 The pNBV (New BiFC Vector) modules. Detailed vector maps for the, A: pNBV gene-YN; B: pNBV gene-YC; C: pNBV YN-gene; D: pNBV YC-gene. YN is the non-fluorescent N-terminal half and YC is the non-fluorescent C-terminal half of YFP. These need to come together for fluorescence to occur (see earlier section). Gene-YN refers to gene fused upstream of YN; YN-gene refers to gene fused downstream of YN. Gene-YC refers to gene fused upstream of YC; YC-gene refers to gene fused downstream of YC.

2.14.3 Design of primers for directional cloning into BiFC vectors (pNBVs)

Primers were designed based on the ORFs encoding mature PINA and PINB proteins

(without the putative signal peptides) for directional cloning into pNBV vectors, in-

frame and upstream or downstream of the YN and YC fragments. For cloning as gene-

YN or gene-YC fusions, the restriction site for XbaI was incorporated at the 5’ end of

the forward primers and the site for XmaI at the 5’ end of reverse primers. For cloning

as YN-gene or YC-gene fusions, the site for EcoRI was incorporated into the forward

primers and for BamHI into the reverse primers. For all forward primers the start codon

was added to Pin sequence. The native stop codon was deleted in the reverse primers

for cloning into two pNBV vectors (gene-YN and gene-YC). The primers listed in

Table 2.11.


- 79 -

Table 2.11 Primers used in directional cloning into pNBV vectors

Primer name Sequence of primers used * Cloning sites Pina-YN

and Pina-YC

Forward: 5’- GGTTCTAGAATGGATGTTGCTGGCGGGGGTG -3’ Reverse: 5’- CCGCCCGGGCCAGTAATAGCCAATAGTGCC -3’

XbaI

XmaI

Pinb-YN and

Pinb-YC

Forward: 5’- GAATCTAGAATGGAAGTTGGCGGAGGAGGTGG -3’ Reverse: 5’- CCGCCCGGGCCAGTAATAGCCACTAGG -3’

XbaI

XmaI

YN-Pina and

YC-Pina

Forward: 5’- GGTGAATTCATGGATGTTGCTGGCGGGGGTG -3’ Reverse: 5’- CCGGGATCCTCACCAGTAATAGCCAATAGTGCC -3’

EcoRI

BamHI

YN-Pinb and

YC-Pinb

Forward: 5’- GAAGAATTCATGGAAGTTGGCGGAGGAGGTGG -3’ Reverse: 5’- CCGGGATCCTCACCAGTAATAGCCACTAGG -3’

EcoRI

BamHI

* Cloning sites to be incorporated for directional cloning are shown in italics and shaded. Nucleotides in italics and wave underline encode start and stop codons. The first residue of mature PINs is boxed. 2.14.4 Restriction enzyme digestion of PCR products

The gene sections encoding the putative mature PINA and PINB, cloned previously

into 3’ viral module vector (pICH11599), were amplified with gene-specific primers

with the appropriate restriction sites and start/stop codons (Table 2.11).

An aliquot of the above Pina and Pinb PCR products was subjected to double-

digestion with XbaI and XmaI.

The four vectors pNBV (gene-YC and gene-YN) and pNBV (YC-gene and YN-

gene) were prepared from their stocks in E. coli Mach1.

The pNBV (gene-YN and gene-YC) were double-digested with XbaI and XmaI.

Four separate ligations were set up, as (i) Pina with pNBV gene-YN vector; (ii)

Pina with pNBV gene-YC; (iii) Pinb with pNBV gene-YN; (iv) Pinb with pNBV

gene-YC.

Another aliquot of the Pina and Pinb PCR products was double-digested with

EcoRI and BamHI.

The pNBV (YN-gene and YC-gene) were double-digested with EcoRI and BamHI.

Four more ligations were set up, as (v) Pina with pNBV YN-gene; (vi) Pina with

pNBV YC-gene; (vii) Pinb with pNBV YN-gene; (viii) Pinb with pNBV YC-gene.

All ligations were transformed into chemically competent cells of E. coli Mach1,

and plated on LB agar supplemented with carbenicillin.


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Several half-colonies per transformation were subjected to colony-PCR to identify

those containing recombinant plasmids.

The half-colonies of interest were cultured and plasmids were prepared from these

using HiYield™ plasmid mini kit and sequenced.

The eight BiFC constructs were sequenced and the correct ligations and ORFs

(pNBV-YN-PINA, pNBV-YC-PINA, pNBV-PINA-YN, pNBV-PINA-YC, pNBV-

YN-PINB, pNBV-YC-PINB, pNBV-PINB-YN and pNBV-PINB-YC) were

confirmed.

2.14.5 Cloning of PIN-YFP fragments into pMLBART vector

The pMLBART vector (Figure 2.10) was kindly provided by Professor David Smyth

and Mr Tezz Quon (Monash University, Melbourne). It is commonly used as a plant

transformation vector for study of protein interactions, by using the NotI restriction site

for cloning and spectinomycin resistance as the selection marker.

Figure 2.10 Plasmid map of pMLBART. Plant transformation vector; NotI site show in red.

All eight BiFC constructs made as above were digested at the NotI site, to release the

section containing the 35S promoter of CaMV, the non-fluorescent N-terminal half of

YFP (YN) or the non-fluorescent C-terminal half of YFP (YC), PINA or PINB,

terminator of the Nos gene (NosT) and LacZ (see Figure 2.9). The inserts were cloned

at the NotI site of pMLBART, the ligation mixtures transformed into E. coli Mach1 and

spread onto LB plates containing 50 μg/mL spectinomycin as well as 0.5 mM IPTG and

pMLBART estimate

9777bp


- 81 -

80 μg/mL X-gal for ‘Blue-white screening’, (based on the principle of α-

complementation of the β-galactosidase (LacZ) gene) which were incubated at 37°C for

16 hours. The white colonies (potentially containing recombinant clones) were tested

by colony PCR. The plasmids were prepared from cultures of insert using the

HiYield™ plasmid mini kit, the inserts confirmed by PCR again and glycerol stocks of

clones prepared and stored at -80°C. The pNBV vector without a Pin insert was also

digested at the NotI site, transformed into E. coli Mach1 cell and the plasmid isolated

(to be used as negative control).

2.14.6 Electroporation into A. tumefaciens GV3101

One µL each of the plasmid DNAs of all eight clones and the negative control in the

pMLBART were each electroporated into A. tumefaciens GV3101 (Section 2.9.7)

grown on LB plates supplemented with 50 μg/mL spectinomycin and 50 µg/mL

rifampicin for two days at 28°C. The colonies were screened by PCR for the presence

of the inserted gene.

2.14.7 Use of TBSV-P19 vector

TBSV-P19 is based on co-expression of a viral suppressor protein of the Tomato Bushy

Stunt virus (TBSV-P19) called P19, that is able to prevent post-transcriptional gene

silencing (PTGS); hence the vector allows high levels of transient gene expression

(Voinnet et al., 2003) (Figure 2.11). It encodes kanamycin resistance and was

transformed previously into Agrobacterium strain LB4404 and stored as a glycerol

stock at -80°C (culture donated by Dr Diane Webster, Monash University).

Figure 2.11 Plasmid map of the P19 vector.


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2.14.8 Agroinfiltration of N. benthamiana using BiFC vectors

The cultures of the eight clones in pMLBART in A. tumefaciens were grown overnight

at 28°C in LB supplemented with 50 μg/mL spectinomycin and 50 µg/mL rifampicin.

Agrobacterium tumefaciens LB4404 containing the P19 vector (see above) was grown

in LB medium supplemented with 50 μg/mL kanamycin and 50 µg/mL rifampicin.

Agrobacterium tumefaciens cultures of various combinations of one pMLBART-YN,

with one pMLBART-YC, and the P19 (all at OD600 of 1) were mixed in a ratio of

1:1:0.5 then the mixture centrifuged at 600×g for 10 minutes at RT. The mixed

bacterial pellet was resuspended in 10 mL of infiltration buffer (Table 2.4) and allowed

to stand for one hour at RT. The suspensions were infiltrated into leaves of 8 week old

N. benthamiana plants using a 1 mL syringe (without a needle) until the entire leaf air

space was filled with the Agrobacterium suspension. Plants were allowed to grow for a

further 3 days under greenhouse conditions before the leaves were harvested and

analysed by fluorescence microscopy.

2.14.9 Fluorescence microscope and imaging

Small sections from the infiltrated area of N. benthamiana leaves were removed and

placed in distilled water on glass slides and covered with cover slips. The lower leaf

epidermis was prepared for higher resolution of the intracellular distribution of any

expressed fluorescence. The samples were examined using ZEISS fluorescence

microscope equipped with AxioCam ICc3 (ZEISS) using a 460-480 nm excitation filter

and a 495-540 nm barrier filter. Images were captured using the AxioVision LE

software (ZEISS) and processed using Adobe Photoshop CS4 software.

2.14.10 Protein methods for detection of BiFC

Total soluble proteins (TSP) were isolated as described earlier (Section 2.13.7) from the

Agrobacterium-infiltrated areas of N. benthamiana leaves potentially expressing the

respective fusion proteins. Protein concentrations were measured by the Bradford

assay. The TSP samples were ultra-filtered and concentrated using an Amicon ultra 0.5

centrifugal filter device (3K) (Millipore, Australia) at 13,000×g and used for western

blots.


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2.14.11 Native gel electrophoresis and detection of YFP fluorescence

The protocol followed Morell et al. (2007) with minor modifications. The TSP

fractions were electrophoresed at 4°C using a 15% ReadyGel® Tris-HCl gel (Bio-Rad,

USA) without SDS in any buffer. The gel was exposed to UV light using Spectroline

UV transilluminator to detect YFP fluorescence.

The overall strategy for construction of BiFC vectors and infiltration into plant cells is

summarized in Figure 2.12.


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Figure 2.12 Strategy used to construction of BiFC vectors for investigating potential interactions of PIN proteins.

Putative PINA cloned in BiFC vectors (pNBV)

Putative PINB cloned in BiFC vector (pNBV)

The BiFC cassettes in pNBV (contains: 35S; YN or YC; PINB; NosT; LacZ) were released from pNBV by NotI digestion to cloned into the plant expression vector, pMLBART

Colony PCR and sequencing

Transformation into A. tumefaciens GV3101

Mixed cultures with bacteria containing P19 were infiltrated into N. benthamiana

ICON vectors used as template DNA for PCR

Four pNBV constructs prepared: PINA-YN/PINA-YC/YN-PINA/YC-PINA

Four pNBV constructs prepared: PINB-YN/PINB-YC/YN-PINB/YC-PINB

The BiFC cassettes in pNBV (contains: 35S; YN or YC; PINA; NosT; LacZ) were released from pNBV by NotI digestion to cloned into the plant expression vector, pMLBART

ICON vector (pICH11599) with putative mature PINB

cloned into MCS

ICON vector (pICH11599) with putative mature PINA

cloned into MCS


- 85 -

2.15 Identification of protein-protein interaction regions of PINs using BiFC

2.15.1 Gifts of gene construct containing deletions of the tryptophan-rich domain

(TRD) of PINA and the hydrophobic domain (HD) of PINA and PINB

The clones designated PINA∆TRD (deletion of TRD at position 34-46 in the mature

PINA protein; Figure 2.13A), PINA∆HD (deletion of HD at position 75-85 in the

mature PINA protein; Figure 2.13B) and PINB∆HD (deletion of HD at position 75-83

in the mature PINB protein; Figure 2.13C) were constructed previously in the pGBK

yeast 2-hybrid system vectors by Dr Rebecca Alfred using the QuikChange II site-

directed mutagenesis kit (Stratagene, California, USA) to delete the above gene sections

(Alfred, 2013 and Alfred et al., 2014). The primers used for introducing the above

deletions are listed in Appendix I (Table I-1). These clones were kindly provided by Dr

Alfred for the present in planta work.

Figure 2.13 Alignment of putative PINA and PINB. A: The tryptophan-rich domain (TRD) highlighted in pink (residues position 34-46) for PINA; B: The hydrophobic domain (HD) highlighted in blue (residues position 75-85) for PINA; C: The hydrophobic domain (HD) highlighted in blue (residues position 75-83) for PINB.

A

B

C


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2.15.2 Cloning of PINA∆TRD and PINA∆HD and PINB∆HD into pNBV vectors

The PINA∆HD, PINB∆HD and PINA∆TRD-encoding genes cloned into pGBK were

used as templates in a second-round PCR (with primers detailed in Table 2.11) to enable

cloning into pNBV for interaction studies by BiFC assay.

Second-round PCR products were sequenced and then purified using HiYield™

gel/PCR fragments extraction kit.

The purified PCR products were double-digested with XbaI and XmaI.

The vector pNBV (gene-YC) were double-digested with XbaI and XmaI.

Two ligations were set up, as (i) PINA-∆HD with pNBV gene-YC (ii) PINA-∆TRD

with pNBV gene-YC.

The purified PCR product for PINB∆HD and the vector pNBV (YN-gene and YC-

gene) were double-digested with EcoRI and BamHI.

Two more ligations were set up, as (iii) PINB-∆HD with pNBV YN-gene (iv)

PINB-∆HD with pNBV YC-gene.

The ligations were transformed into E. coli Mach1 and plated on LB agar

supplemented with carbenicillin.

Several colonies per transformation were subjected to colony-PCR to identify those

containing recombinant plasmids.

The plasmids were prepared from cultures of this using HiYield™ plasmid mini kit

and sequenced.

The four PIN mutant BiFC constructs made as above were digested at the NotI site

and cloned into the NotI site of pMLBART. The ligation mixtures were

transformed into E. coli Mach1 and spread onto LB plates containing 50 μg/mL

spectinomycin, 0.5 mM IPTG and 80 μg/mL X-gal for ‘Blue-white screening’.

The white colonies (containing recombinant clones) were tested by colony PCR.

The plasmids were prepared from cultures of this using HiYield™ plasmid mini kit

and the inserts were confirmed by PCR again and glycerol stock of clones were

prepared and stored at -80°C. All further steps were as per the sections 2.14.6 and

2.14.8


- 87 -

2.15.3 Cloning of PINA∆TRD and PINA∆HD and PINB∆HD into the 3’ module

pICH11599

The above-made clones in pNBV (encoding PINA∆TRD, PINA∆HD and PINB∆HD)

were used as template in a second-round PCR with gene specific primers with the

appropriate restriction enzyme sites for pICH11599 (Table 2.7). Second-round PCR

products were purified using HiYield™ gel/PCR fragments extraction kit, double-

digested with appropriated restriction sites and ligated with the digested pICH11599

(Figure 2.14). The ligations were transformed into E. coli Mach1 and plated on LB agar

supplemented with carbenicillin. Several colonies per transformation were subjected to

colony-PCR to identify those containing recombinant plasmids. The plasmids were

prepared from cultures of E. coli using HiYield™ plasmid mini kit and sequenced. All

further steps were as per sections 2.13.4 and 2.13.5.

Figure 2.14 Generation of the mutant PIN constructs for magnICON® viral vectors. A: the region of PINA∆HD and PINB∆HD, encoding the PIN proteins without HD, followed by the His-tag sequence, ligated into; B: the magnICON® viral vector 3’ module; C: the region of PINA∆TRD encoding the PIN proteins without TRD ligated into magnICON® viral vector 3’ module.

CHAPTER 3

Expression, purification and antimicrobial activity of PINA and PINB proteins in Nicotiana benthamiana

Chapter 3 Protein expression

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3.1 Introduction

Protein expression and purification is required in a number of biochemical

investigations such as protein identifications, sequencing, testing of functionality

including enzymatic activities and three dimensional structure determinations.

Therefore, transgenic plant technology, followed by biochemical and techniques, were

applied to PIN proteins to understand the roles they play in determining grain texture,

protection against pathogens and the mechanisms of action in both aspects. For the past

two decades, industrially and therapeutically important proteins have been successfully

expressed in plant systems. However, low yield and lack of efficient purification

methods are two major challenges for economical production of plant-made

recombinant proteins (Conley et al., 2011). Further biochemical characterisation of the

PIN proteins such as three-dimensional structure by X-ray crystallography is essential to

better understand their properties and functions. PINs could be an alternative to

conventional antibiotics to control plant and human diseases due to their antimicrobial

properties. Despite several investigations, there are no reports of PIN proteins purified

from wheat in high yield. However, a small number of studies have identified the lipid-

binding nature of PIN extracted and purified directly from wheat flour (Clifton et al.,

2007b; Clifton et al., 2008; Clifton et al., 2011b). Recombinant PINs have also been

purified from E. coli based expression systems, with varying degrees of success

(Capparelli et al., 2006; 2007; Miao et al., 2012). In addition, Sorrentino et al. (2009)

showed that PINB can be produced in transgenic tobacco cells, targeted to chloroplast,

with the maximum yield of only 0.35% of the total soluble proteins (TSP). In all these

cases the purpose of producing the recombinant PINs was to assess their antimicrobial

activity (detailed in Chapter 1).

The present work aimed to clone and transiently over-express PINA and PINB in

planta, using expression systems based on plant viral vectors. The magnICON® system

was used to produce C-terminal His-tagged fusion proteins in the cytosol, chloroplast,

apoplast and ER. In addition, various strategies were tested for optimising the

production of recombinant PIN within the TSP fraction, including the time of harvest

and extraction buffers. It was necessary to subsequently purify the proteins to avoid

effects of plant-based alkaloids during antimicrobial assays. Two purification systems


- 90 -

based on His-tag affinity and hydrophobic interactions, were used. To investigate the

functionality of the in planta expressed PINs, antibacterial and antifungal assays were

then performed.

The results in this chapter are divided into 3 sections: expression, purification and

characterisation of the antimicrobial activity of the PINA and PINB.


- 91 -

600bp 400bp

400bp 200bp

200bp

3.2 Expression of PINs using magnICON® viral vectors

3.2.1 Cloning PINA and PINB into magnICON® viral vector system

The 447 bp genes encoding the full-length putative PINA and PINB proteins were first

amplified from the gDNA of the soft wheat line Triticum aestivum cv. Rosella using the

primers given in Table 2.6 (results not shown). For directional cloning into the 3’

module of the magnICON® system, i.e., pICH11599, these products were used as

templates in second-round PCR (using primers listed in Table 2.7) to amplify the gene

sections encoding the putative mature PINA and PINB with C-terminal His-tags, or His-

SEKDEL-tags, and appropriate restriction sites. PCR products of ~400 bp obtained for

PINA-His and PINB-His (Figure 3.1) were consistent with sizes of the gene sections

encoding start codon (3 bp), the putative mature PINA (363 bp) or PINB (360 bp), and

the 6×His tag (18 bp) (Table 2.8). PCR products of ~400 bp were also obtained for

mature PINA-His-SEKDEL and mature PINB-His-SEKDEL, consistent with the gene

sections encoding mature PINA (363 bp) or PINB (360 bp) and the added start codon (3

bp) and tags (6×His, 18 bp; and SEKDEL, 18 bp) (data not shown).

Figure 3.1 Second-round PCR of gene sections encoding mature PINs. Lane 1: Negative control (no template); lane 2: PINA-His (mature protein-encoding section of Pina-D1a with His-tag); lane 3: PINB-His (mature protein-encoding section of Pinb-D1a with His-tag); lane M: DNA molecular weight marker, Hyperladder l. As per the primer design (Section 2.13.2), the PINA-His and PINA-His-SEKDEL PCR

products and the vector pICH11599 were double-digested with EcoRI+BamH1, for

ligations. The PINB-His PCR products were double-digested with NcoI+SacI and

PINB-His-SEKDEL PCR products with EcoRI+BamH1, and ligated with similarly-

1 2 3 M


- 92 -

digested pICH11599. The four ligations were transformed into E. coli Mach1 cells.

Due to lack of blue/white colour-selection (lacZ) on pICH11599, colony PCR was

carried out using PIN primers (Table 2.7) to test for inserts (approximately 400 bp), as

exemplified in Figure 3.2. Plasmids were isolated from the above clones and double-

digested with the appropriate enzyme combination, to also ensure the presence of

correct-sized inserts. The results are exemplified in Figure 3.3.

Figure 3.2 Colony PCR for preliminary selection of clones. Lanes M: marker, Hyperladder 1; lanes 1-4: pICH-PINA-His products; lane 5-8: pICH-PINB-His products; lane 9: negative control (no template); lanes 10-11: pICH-PINA-His-SEKDEL products; lanes 12-13: pICH-PINB-His-SEKDEL.

Figure 3.3 Example of double digests of clones in pICH11599. Lane M: marker, Hyperladder 1; lanes 1, 2: undigested and EcoRI+BamHI digested pICH-PINA-His clone respectively; lanes 3, 4: undigested and NcoI+SacI digested pICH-PINB-His clone respectively; lanes 5, 6: undigested and EcoRI+BamHI digested pICH-PINA-His-SEKDEL clone respectively; lanes 7, 8: undigested and EcoRI+BamHI digested pICH-PINB-His-SEKDEL clone.

M 1 2 3 4 5 6 7 8


- 93 -

For each construct, four potential clones were sequenced using gene-specific primers

and alignments of the data with the published sequences, Pina-D1a (DQ363911) and

Pinb-D1a (DQ363913) showed high identity with the corresponding sections. The

sequences are provided in Appendix II (Figure II-1 to II-4). The constructs were

designated pICH-PINA-His; pICH-PINB-His; pICH-PINA-His-SEKDEL; pICH-PINB-

His-SEKDEL (Figure 3.4). Each construct was electroporated into A. tumefaciens

GV3101 (Section 2.13.4). Screenings of clones containing inserts were carried out

using colony-PCR as above (data not shown). The various 5’ modules, the 3’ GFP

module and the integrase module (Section 2.13.1) were also transformed individually

into A. tumefaciens.

Figure 3.4 The 3’ module constructs encoding mature PIN with His-tag and His/SEKDEL tags. A: pICH-PINA-His; B: pICH-PINB-His; C: pICH-PINA-His-SEKDEL; D: pICH-PINB-His-SEKDEL.

His Tnos NPT Pnos Intron 3’NTR Tnos AttB

RB LB EcoRI BamHI

PINA

His PINB Tnos NPT Pnos Intron 3’NTR Tnos AttB

RB LB NcoI SacI

PINA His

SEKDEL EcoRI

Tnos NPT Pnos Intron 3’NTR Tnos AttB

RB LB EcoRI BamHI EcoRI

PINB His

SEKDEL EcoRI

Tnos NPT Pnos Intron 3’NTR Tnos AttB

RB LB EcoRI BamHI

A

B

C

D


- 94 -

3.2.2 Transient expression of PIN proteins in the viral vector system

The A. tumefaciens transformants were used in combinations of three cultures, one with

a 5’ module (for targeting the expressed protein to different cellular compartments), one

with a 3’ module (empty, or with PIN or GFP inserts), and one with the integrase

module to allow recombination of the former two modules and hence recombinant

protein expression. The cytosol and chloroplast targeting GFP served as positive

control, while cultures of the 5’ cytosol and the integrase modules (and no 3’ module)

served as negative control. The combinations are detailed in section 2.13.5 (Table 2.9).

The cultures were mixed, pelleted, resuspended and infiltrated into the underside of N.

benthamiana leaves for transiently expressing the encoded proteins. Leaves were

harvested from three independent plants for each agro-infiltration, before and after

necrosis developed in that area. The necrosis suggested protein production, as the

negative control (5’+integrase modules only) developed marginal necrosis (Figure 3.5).

A B Figure 3.5 N. benthamiana leaves 10 days post-infiltration (different leaves on one plant). A: agro-infiltration with the 5’ cytosol+integrase modules only (negative control); B: agro-infiltration with 3’ (pICH-PINB-His)+integrase+cytosol modules. Agro-infiltrated areas are shown by arrows and dotted lines.

The total soluble protein (TSP) was extracted (Section 2.13.7) from each infiltration

area, GFP expression tested, and western blot and ELISA applied for PINs. Four

different batches were tested and extracts with the highest amounts of PINA or PINB

were preferentially used for further purification and test activity. The results are

detailed below.


- 95 -

Chloroplast Cytosol

3.2.3 Expression of Green Fluorescent Protein (GFP) by the viral vector system

GFP is widely used as a reporter for gene expression and localisation (Ward, 2006), as

an in situ tag for fusion proteins (Cremazy et al., 2005; Wang and Hazelrigg, 1994), a

biosensor (Crone et al., 2013; Lalonde et al., 2005) or a probe for protein-protein

interactions (Magliery and Regan, 2006; Ventura, 2011). It is 238 amino acids long

(26.9 kDa) and forms a ß-barrel around one α-helix (Ormö et al., 1996; Yang et al.,

1996). GFP expression (Section 2.13.6) was used as a positive control to ensure the

viral vector-based heterologous expression was working correctly. In conjunction with

the 3’ GFP module and integrase, two separate 5’ modules (for cytosol or chloroplast),

were agro-infiltrated and production of GFP was observed over time, between 10-14

days post infiltration (dpi). Leaves were examined using UV light and GFP protein

showed bright fluorescence (Figure 3.6).

A Chloroplast B Cytosol

Figure 3.6 GFP production using two different targeting modules, in N. benthamiana leaves (10 dpi). A: Agro-infiltrated leaves viewed under natural light; B: GFP production in chloroplast and cytosol viewed under UV. The TSP extracted from infiltrated leaves using 3’ GFP module and different 5’

modules (cytosol or chloroplast) were quantified using Bradford assay. The absorbance

of each sample was read at 595 nm and the TSP concentration determined from the

standard curve (Figure 3.7), constructed using bovine serum albumin (BSA) standards

ranging from 0.1 to 0.5 mg/mL (Section 2.11.1). Table 3.1 shows the concentration of

TSP from leaves infiltrated with chloroplast-targeted GFP, cytosol-targeted GFP and

also negative controls. Approximately 93 µg and 98 µg of the quantified TSP samples

were run on a gel to analyse the GFP expression by SDS-PAGE gel (data not shown)

and western blot. Western blot analysis using anti-GFP antibody (Section 2.3)

confirmed the presence of GFP at approximately 25 kDa, at the expected size for GFP

(Figure 3.8).


- 96 -

Figure 3.7 Standard curve of BSA concentration for Bradford assay. The standard curve was prepared using BSA in PBS buffer, and absorbance measured at 595 nm. Table 3.1 Quantification of TSP for targeted expression of GFP using Bradford assay

Protein Samples Average of test OD595

Protein calculation

based on standard curve (mg/mL)

Dilution factor

Amount of TSP

(mg/mL)

TSP loaded on SDS (µL)

Protein on gel (µg)

GFP Cytosol 0.698 ± 0.005 0.264 10 2.64 37 97.68 GFP Chloroplast 0.685 ± 0.004 0.252 10 2.52 37 93.24 Negative Cytosol 0.594 ± 0.007 0.168 10 1.68 37 62.16 Negative Chloroplast 0.577 ± 0.005 0.152 10 1.52 37 56.24

Figure 3.8 Western blot analysis to determine GFP expression using anti-GFP antibody. Lane 1: TSP from leaf infiltrated with 5’ chloroplast+integrase modules only (negative control); lane 2: TSP from leaf infiltrated with 5’ chloroplast+integrase+3’ GFP modules; lane 3: void lane (no sample loaded); lane 4: TSP from leaf infiltrated with 5’ cytosol+integrase+3’ GFP modules; lane 5: TSP from leaf infiltrated with 5’ cytosol+integrase modules only (negative control).

y = 1.0865x + 0.4124 R² = 0.9843

00.20.40.60.8

11.2

0 0.1 0.2 0.3 0.4 0.5 0.6Abs

orba

nce

(595

nm

) BSA concentration (mg/mL)

1 2 3 4 5

25kDa

93µg 98µg

37kDa

75kDa

56µg 62µg


- 97 -

3.2.4 Expression of the recombinant PINA protein by the viral vector system

Following agro-infiltration with pICH-PINA-His and pICH-PINA-His-SEKDEL using

different 5’ vector modules, leaves were harvested. The TSP extracted from these were

quantitated by Bradford assay (Table 3.2), then analysed by SDS-PAGE and Coomassie

Blue staining of gels (Figure 3.9). The results showed some major bands at ~50 kDa

and ~15 kDa as well as some minor bands (e.g., 75 kDa, 37 kDa and 25 kDa). Since the

GFP production showed the system was functioning as expected (Figure 3.6), PIN

proteins were then detected by western blot using the Durotest, anti-friabilin

monoclonal antibody (described in Section 2.11.3).

Figure 3.9 Coomassie Blue stained gel to analyse TSP from infiltrated leaves. Lane 1: infiltration with chloroplast targeted PINA; lane 2: infiltration with cytosol targeted PINA; lane 3: infiltration with apoplast targeted PINA; lane 4: infiltration with ER targeted PINA.

The molecular weights of the C-terminally His-tagged PINA, with and without

SEKDEL, were estimated at 14.97 kDa and 14.27 kDa respectively, using the ‘compute

pI/Mw tool’ at the ExPASy (Section 2.10.3). The predicted subcellular localisation of

PINA by WoLF PSORT (Section 2.10.4) showed the highest score for chloroplast

targeting. The PIN proteins were detected by western blot using the Durotest antibody.

The positive control supplied with the Durotest kit (3% non-durum wheat) exhibited a

band at ~15 kDa (Figure 3.10; lane 1), the expected size for the friabilin protein mixture

composed mainly of PINA and PINB, originating from the non-durum wheat.

1 2 3 4

75kDa

50kDa

37kDa

15kDa

25kDa


- 98 -

Interestingly, the recombinant PINA was detected at the unexpected size of ~45 kDa

(between 50 kDa and 37 kDa) when targeted to different parts of the plant cell, i.e.,

chloroplast, cytosol and ER, but not detected in the apoplast, probably due to limited

protein delivery to this compartment. Further, the ~45 kDa bands were observed only

when the TSP samples were not heated prior to the western blot, and could not be

detected when the TSP samples were heated (as discussed later in section 3.2.6).

Figure 3.10 Determination of PINA expression by western blotting of TSP extracts from infiltrated leaves. Lane 1: 3% non-durum wheat as positive control for antibody; lane 2: negative control (5’+ integrase modules only); lane 3: cytosol targeted PINA; lane 4: apoplast targeted PINA; lane 5: chloroplast targeted PINA; lane 6: ER targeted PINA. The quantified TSP samples, as well as the negative controls, were used for enzyme-

linked immunosorbent assay (ELISA) analysis (Section 2.11.4) to measure the yield of

recombinant PINA in N. benthamiana leaves. High-binding ELISA plates were coated

with 0.3 µg TSP/well. The assay tested protein samples in three different dilutions

(1/50, 1/100 and 1/200). The absorbance of each sample was read at 450 nm, and the

PINA concentration determined from the 3% non-durum wheat (supplied in the

Durotest kit) standard curve, ranging from 0.0675-0.54 µg/mL (Figure 3.11).

1 2 3 4 5 6

50kDa

15kDa

75kDa

37kDa


- 99 -

Figure 3.11 Standard curve of 3% non-durum wheat dilutions for ELISA assay.

Table 3.2 shows the concentration of the recombinant PINA (rPINA) in infiltrated

leaves for the 1/200 dilution test. The maximum yield obtained for PINA was ~0.836%

of TSP, or approximately ~51.7 µg/g of fresh weight using the 5’ module for

chloroplast (Figure 3.12). The PINA yields were ~0.642% TSP or ~40.5 µg/g of fresh

weight using the 5’ module for cytosol and ~0.63% TSP or ~39.7 µg/g of fresh weight

for ER.

Table 3.2 ELISA data analysis for quantification of recombinant PINA (rPINA) protein Sample name OD450

a

rPINA (µg/mL)

rPINA (mg/mL)b

TSP (mg/mL)

FWc (µg/g)

Yield of rPINA (%)

PINA-Chloroplast 0.663 ± 0.0126 5.173 0.00517 0.702 ± 0.059 51.7 0.836 ± 0.033

PINA-Cytosol 0.658 ± 0.010 4.052 0.00405 0.602 ± 0.029 40.5 0.642 ± 0.032 PINA-ER 0.667 ± 0.012 3.972 0.00397 0.612 ± 0.044 39.7 0.63 ± 0.033 PINA-Apoplastd 0.10877 ± 0.003 - - - - - Negatived 0.0547 ± 0.0015 - - - - - a average of at least 4 independent tests; b different dilutions were tested, data show 200-fold dilution. c FW: fresh weight; d data not applicable for apoplast and negative samples.

C h loro

p las t

C y tos o l

E R0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

P I N A

T a rg e tin g

Per

cen

tag

e

Figure 3.12 Quantification of PINA proteins targeted to chloroplast, cytosol and ER by ELISA.TSP extract from leaves harvested after infiltration was added to ELISA plate and PINA detected using the Durotest antibody. Bars show the mean percentage of protein extracts from four individual leaves and the error bars indicate the standard error of the mean (SEM).

y = 1.6016x-1.044 R² = 0.9927

0

0.2

0.4

0.6

0.8

1

1.2

0 0.1 0.2 0.3 0.4 0.5 0.6

Abs

orba

nce

(450

nm

)

3% non-durum wheat concentration (µg/mL)

0.836%

0.642% 0.63%


- 100 -

3.2.5 Expression of the recombinant PINB protein by the viral vector system

The constructs pICH-PINB-His and pICH-PINB-His-SEKDEL made using different 5’

vector modules were infiltrated into N. benthamiana leaves. The TSPs were extracted

and analysed using SDS-PAGE (Figure 3.13) and quantitated by Bradford assay (Table

3.3).

Figure 3.13 Coomassie Blue stained gel to analysis TSP from infiltrated leaves. Lane 1: infiltration with chloroplast targeted PINB; lane 2: infiltration with cytosol targeted PINB; lane 3: infiltration with ER targeted PINB; lane 4: infiltration with apoplast targeted PINB.

The recombinant PINB (rPINB) was detected by western blot using the above antibody.

The molecular weights for C-terminally His-tagged PINB with or without the SEKDEL

were predicted, using the ‘compute pI/Mw tool’ at the ExPASy (Section 2.10.3) at

15.13 kDa and 14.43 kDa, respectively. The predicted subcellular localisation of PINB

by WoLF PSORT (Section 2.10.4) showed the highest score for chloroplast targeting.

The western blot showed the friabilin proteins at the expected size (~15 kDa), in the

positive control, 3% non-durum wheat. The recombinant PINB with or without the

SEKDEL was detected at the unexpected size of ~45 kDa bands (between 50 kDa and

37 kDa) (Figure 3.14), only when the TSP samples were not heated prior to western blot

(as discussed in section 3.2.6). The ~45 kDa PINB was noted when targeted to the

chloroplast, cytosol or ER, but not when targeted to apoplast.

1 2 3 4

75kDa

50kDa

37kDa

15kDa

25kDa


- 101 -

Figure 3.14 Determination of PINB expressions by western blotting of TSP extracts from infiltrated leaves. Lane 1: 3% non-durum wheat as positive control for antibody; lane 2: negative control (5’+integrase modules only); lanes 3 and 9: ER targeted PINB; lanes 4 and 5: cytosol targeted PINB; lanes 6 and 7: chloroplast targeted PINB lane 8: apoplast targeted.

The TSP samples from freshly infiltrated leaves were quantified by Bradford assay and

analysed by ELISA (Section 2.11.4) with Durotest antibody to measure the yield of

recombinant PINB protein. ELISA plates were coated with 0.3 µg TSP/well. The assay

tested protein samples in three dilutions (1/50, 1/100 and 1/200). The absorbance was

read at 450 nm, and the PINB concentration determined from the 3% non-durum wheat

standard curve as above (Figure 3.11). Table 3.3 shows the concentration of PINB in

infiltrated leaves in 1/200 dilution test. As shown in Figure 3.15, the maximum yield

for PINB was ~0.894% of TSP, or approximately ~70 µg/g of fresh weight using the 5’

vector module for chloroplast. PINB yields of up to ~0.73% and ~0.68% TSP, or ~45

µg/g and ~40 µg/g of fresh weight were obtained for cytosol and ER targeting,

respectively.

Table 3.3 ELISA data analysis for quantification of recombinant PINB (rPINB) protein

Sample name OD450a

rPINB (µg/m

L)

rPINB (mg/mL)b

TSP (mg/mL)

FWc (µg/g)

Yield of rPINB (%)

PINB-Chloroplast 0.66 ± 0.011 6.294 0.00629 0.762 ± 0.045 62.9 0.894 ± 0.051 PINB-Cytosol 0.661 ± 0.012 4.533 0.00453 0.692 ± 0.060 45.3 0.734 ± 0.028 PINB-ER 0.66 ± 0.019 4.053 0.00405 0.615 ± 0.034 40.5 0.672 ± 0.023 PINB-Apoplastd 0.113 ± 0.0056 - - - - - Negatived 0.0547 ± 0.001 - - - - -

a average of at least 4 independent test; b different dilutions were tested, data show 200-fold dilution; c FW: fresh weight; d data not applicable for apoplast and negative samples.

1 2 3 4 5 6 7 8 9

50kDa

15kDa

75kDa

37kDa


- 102 -

C h loro

p las t

C y tos o l

E R0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

P IN B

T a rg e tin g

Per

cent

age

Figure 3.15 Quantification of PINB proteins targeted to chloroplast, cytosol and ER by ELISA. TSP extract from leaves harvested after infiltration was added to ELISA plate and PINB detected using the Durotest antibody. Bars show the mean percentage of protein extracts from four individual leaves and the error bars indicate the standard error of the mean (SEM). 3.2.6 Optimisations of expression of His-tagged recombinant PINA and PINB

The optimal time of PIN harvest was investigated by infiltrating leaves with constructs

for targeting to the chloroplast and cytosol and harvesting at 8, 10 or 12 days post-

infiltration (dpi). One µg of TSP (as assayed by Bradford method) was used for ELISA

in two dilutions (1/100 and 1/200). Total soluble protein (TSP) from the infiltrated

leaves harvested at 10 dpi resulted in a slightly higher OD450 compared to 8 or 12 dpi

(Figure 3.16). Overall, the maximal yields of PINs were achieved at 10 dpi, using these

modules.

D a y 8

D a y 10

D a y 12

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C y to s o l

H a rv e s t

OD

450n

m

P IN A

P IN B

N eg

D a y 8

D a y 10

D a y 12

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C h lo r o p la s t

H a rv e s t

OD

450n

m

P IN A

P IN B

N eg

Figure 3.16 Detection of PIN proteins harvested at different times post-infiltration, by ELISA. Quantified TSP samples were added to ELISA plate and PINs detected using the Durotest antibody. The negative control was protein extracted from leaf material infiltrated with only the 5’+integrase modules. The bars indicate the mean OD450 of protein extracts from two individual leaves and the error bars indicate the standard error of the mean (SEM).

0.894%

0.73% 0.68%


- 103 -

The quantified TSP samples (approximately 50 µg) were heated at 60°C, 65°C and

70°C for 15, 30 seconds and 1 minute to denature any recombinant PINs prior to

western blotting. No bands were observed at 70°C for the tested proteins at all three

time points (data not shown). The same result was observed for 60°C and 65°C as

shown in Figure 3.17. Heating at 65°C for 30 seconds led to faint bands with size shift

near 37 kDa compared to the unheated samples (Figure 3.17). No bands could be

detected when the samples were heated at 65°C for 1 minute (Figure 3.17). This

demonstrates that the plant-made recombinant PINs are heat-sensitive.

Figure 3.17 Heat sensitivity of plant-expressed PINA and PINB detected by western blot. Lane 1 and 2: chloroplast-targeted PINA and PINB, respectively, in TSP heated at 65°C for 30 seconds; lanes 3 and 4: these PINA and PINB in TSP heated at 65°C for one minute; lane 5 and 6: these PINA and PINB, in TSP not heated; lane 7: negative control (5’+integrase modules only).

1 2 3 4 5 6 7

50kDa

15kDa

75kDa

37kDa


- 104 -

A number of different extraction buffers (Section 2.13.7; Table 2.10) were then tested

and the proteins detected by western blot. Extraction buffer 1 was used for all further

analyses, as other buffers did not improve the yield or extent of denaturation of PINs.

Further, in the optimisations, only one western blot confirmed the chloroplast-targeted

PINA and PINB at ~15 kDa (Figure 3.18), expected for His-tagged PINA and PINB

monomers, predicted to be 14.27 kDa and 14.43 kDa, respectively. These bands were

obtained using extraction buffer 1 which was without EDTA, and without heating the

TSP, and when approximately 90 µg of TSP samples were loaded on gels.

Figure 3.18 Determination of PINA and PINB protein expression in infiltrated leaves with viral vector module by western blot of TSP in expected size. Lane 1: chloroplast targeted PINA (~15 kDa); lane 2: chloroplast targeted PINB (~15 kDa); lane 3: negative control (5’+integrase modules only); lane 4: 3% non-durum wheat as positive control.

1 2 3 4

25kDa

15kDa

37kDa

50kDa


- 105 -

3.3 Results for purification of PINA and PINB

3.3.1 His-tag purification under native conditions

The PINs were purified from TSP of leaf infiltrated with pICH-PINA-His or pICH-

PINB-His, in combination with the chloroplast 5’ module, due to the high yield of

recombinant PINs when targeted to the chloroplast. Purification was performed using

Ni-NTA resin column (Section 2.13.9). In order to increase the chances of C-terminal

His-tag being correctly folded and functional, the soluble PIN-His proteins were

purified under native conditions (without denaturing buffer) and at 4°C to minimize

protein degradation. Samples (approximately 50 µg) that had indicated chloroplast-

expressed PINs by western blots before purification, and the fractions passed over a Ni-

NTA resin column with wash buffer (Table 2.5), were run on 15% SDS-PAGE and

visualized by Coomassie Blue staining (Figure 3.19). The washed fractions with

imidazole (25 mM) in the wash buffer showed faint bands at 50 kDa. However, the 37

kDa and 15 kDa bands were absent on gel (Figure 3.19; lane 4, 5).

Figure 3.19 SDS-PAGE of TSP and wash steps of His-tag purification under native conditions. Lane 1: Dual Xtra standards (Bio-Rad); lane 2 and 3: TSP of chloroplast-expressed PINA and PINB respectively; lane 4 and 5: fractions from Ni-NTA column with imidazole (25 mM) in wash buffer for PINA and PINB purification respectively.

50

37

15

1 2 3 4 5

75

250

25

kDa


- 106 -

His-tagged PINs were eluted with an elution buffer containing 250 mM imidazole

(Table 2.5) and then dialysed to remove imidazole, then concentrated (Sections 2.13.12

and 2.13.13) and quantitated using Bradford assays. This was followed by SDS-PAGE

and western blot analysis with ~48 µg and ~52 µg for PINA and PINB, respectively

(Figure 3.20). The fractions exhibited a few faint bands and strong bands at 37 kDa and

15 kDa for both PINs in the stained gel (lanes 2, 3) which may correspond to the

multimer and monomer sizes of His-tagged PINs, respectively. The results indicate that

the target proteins were retained on the resin and were only visible after elution with

high imidazole (250 mM) in the elution buffer. The western blot of the eluted fractions

(42 µg, 48 µg and 52 µg protein for negative control, PINA and PINB, respectively)

indicated a broad band at ~37 kDa, confirming the identity of 37 kDa bands as PIN

proteins (Figure 3.20; lanes 6, 7). However, the 15 kDa band not shown signal with

antibody which may not just detected with Durotest. The faint band above ~37 kDa

reacted with the Durotest antibody in PINA and PINB samples (lane 6 and 7) which was

probably observed as 45 kDa protein when TSP samples were analysed.

Figure 3.20 SDS-PAGE and western blot analysis with Durotest antibody for eluted fraction on Ni-NTA resin columns based on His-tag purification under native conditions. Lane 1 and 5: eluted fraction of negative control (5’+integrase modules only); lane 2 and 6: eluted fraction of PINA on SDS-PAGE and western blot respectively; lane 3 and 7: eluted fraction of PINB on SDS-PAGE and western blot respectively; lane 4: Dual Xtra standards (Bio-Rad).

75kDa

1 2 3 4 5 6 7

50kDa

37kDa

15kDa

42µg 48µg 52µg 42µg 48µg 52µg


- 107 -

3.3.2 His-tag purification under denaturing conditions

His-tag purification was performed using the refolding method (Section 2.13.10) to

observe the effect of a protein denaturant such as urea, which disrupts the non-covalent

bonds and increases protein solubility. His-tag purification generally works well under

denaturing conditions (Waugh, 2005). Purification steps were typically performed at

4°C to minimize degradation. PINs were eluted from the Ni-NTA column with a buffer

containing 250 mM imidazole and 6 M urea (Table 2.5) then dialysed (Section 2.13.12)

to remove imidazole and urea, and protein concentrations measured by Bradford

assays. This was followed by SDS-PAGE and western blotting with ~32 µg and ~35

µg for eluted samples of PINA and PINB, respectively, and 30 µg for negative control.

A single band was observed for PINA and PINB at approximately 15 kDa on the gel

(Figure 3.21; lanes 2 and 3) which could be their putative monomer. The same

amounts of protein samples were analysed by western blot, however the denatured

monomeric forms of both PINs were not detected (lanes 6 and 7).

Figure 3.21 SDS-PAGE and western blot analysis with Durotest antibody for eluted fraction on columns with 6M urea in elution buffer. Lane 1: Dual Xtra standards (Bio-Rad); lane 2 and 6: eluted fraction of PINA on SDS-PAGE and western blot respectively; lane 3 and 7: eluted fraction of PINB on SDS-PAGE and western blot respectively; lane 4 and 8: eluted fraction of negative control (5’+integrase modules only) lane 5: 3% non-durum wheat as a positive control for antibody.

30 µg

1 2 3 4 5 6 7 8

37

25

20

15

kDa

32 µg 35 µg 30 µg


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3.3.3 Hydrophobic interaction purification

In a previous attempt to purify the PINs using His-tag, the proteins had not shown a

dominant band on SDS-PAGE (results in section 3.3.1). This may be due to the other

plant proteins binding to the Ni-NTA resin. Therefore, hydrophobic interaction

purification, a separation technique that uses the properties of hydrophobicity to

separate proteins from one another, was applied (Section 2.13.11). Proteins passing

through the column with hydrophobic amino acid side chains on their surfaces are able

to bind to the hydrophobic groups on the column. The interactions are too weak in

water (a polar solvent), however the additions of salts such as ammonium sulfate

[(NH4)2SO4] to the buffer result in stronger hydrophobic interactions. Ammonium

sulfate concentrations of 60%, 70% and 80% saturation were used for phase separation

by ethanol extraction (Figure 3.22).

Figure 3.22 Fractions in the course of extraction procedure. Protein was extracted into upper phase (ethanol) after addition of ammonium sulfate (60%, 70% and 80% saturation) and ethanol.

The proteins were eluted from Butyl-Toyopearl column, quantified by Bradford assay

and ~40 µg of each preparation was run on SDS–PAGE gel and visualized by

Coomassie Blue staining. 70% and 80% ammonium sulfate did not show clear bands of

PINs (data not shown). However, elution with reduced (60%) ammonium sulfate led to

pure proteins, seen as two bands at ~37 kDa and ~15 kDa, assumed to be a multimeric

and monomeric forms of PINs (Figure 3.23; lanes 2 and 3). It is notable that these

bands were only recovered by hydrophobic interaction purification. Further, strong

bands at about 50 kDa were observed on the gel for chloroplast PIN proteins and also

negative control (Figure 3.23; lanes 2, 3, 5). This band may represent the ribulose 1, 5-

60% 70% 80%


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bisphosphate carboxylase/oxygenase (RuBisCO) large subunit, which is the most

abundant protein in leaves (Bally et al., 2009).

Figure 3.23 SDS-PAGE for fractions eluted from the Butyl-Toyopearl column based on hydrophobic interaction system. Lane 1: Dual Xtra standards (Bio-Rad); lane 2 and 3: eluted fractions of PINA and PINB respectively; lane 4: no sample; lane 5: eluted fraction of negative control (5’+integrase modules only). Further purification was attempted in order to remove Rubisco. TSP fractions were

prepared as per previous purifications and passed over a Butyl-Toyopearl column.

After this, RuBisCO was removed using sodium phytate precipitation (Section 2.13.14).

The stained gel showed successful removal of Rubisco, but the bands of interest (37

kDa and 15 kDa) were also very faint (Figure 3.24; lanes 2 and 3).

Figure 3.24 SDS-PAGE of eluted fraction on Butyl-Toyopearl column based on hydrophobic interaction system and sodium phytate precipitation. Lane 1: Dual Xtra standards (Bio-Rad); lane 2 and 3: eluted fractions of PINA and PINB with sodium phytate precipitation respectively.

1 2 3 kDa

50

37

15

75

50

37

15

1 2 3 4 5

75

kDa


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Western blot analysis using the Durotest antibody resulted in a broad band at ~37 kDa,

confirming this as the multimer form of PINA and PINB (Figure 3.25; lanes 1 and 2).

The result confirmed that this antibody is specific for both PINs, but also suggest that it

is most likely not able to recognize the monomer forms of PINA or PINB, as seen

earlier (Figure 3.20).

Figure 3.25 Western blot of eluted fractions on Butyl-Toyopearl column based on hydrophobic interaction system. Lane 1 and 2: eluted fractions of PINA and PINB, respectively; lane 3: eluted fractions of negative control (5’+integrase modules only).

1 2 3

37kDa

25kDa

15kDa


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3.3.4 Western blot analysis using anti His-tag for PIN proteins

The TSP of infiltrated leaves using 3’ PINA and PINB modules and different 5’ vector

modules (cytosol or chloroplast) were subjected to western blot analysis with Durotest

antibody. The samples which showed a signal with this antibody were used for western

blot with anti-His antibody (Table 2.3). Different concentrations of proteins and anti-

His antibody were tested, but no signals detected. Further, after purification with two

different systems, i.e., His-tag purification under native conditions, and hydrophobic

interaction purification, purified PINs were used for western blots. Following His-tag

purification under native conditions, no bands for C-terminally His-tagged PINs were

observed (Figure 3.26; lanes 3 and 4), while the samples after hydrophobic purification

showed two bands, corresponding to the PIN multimer of ~37 kDa and monomer size of

~15 kDa (lanes 1 and 2).

Figure 3.26 Western blot analysis of PIN proteins purified with two different systems. Lane 1 and 2: eluted fractions of PINA and PINB from Butyl-Toyopearl columns respectively; lane 3 and 4: eluted fractions of PINA and PINB on Ni-NTA resin columns respectively; lane 5: eluted fraction of negative control (5’+integrase modules only). After purification the recombinant PIN proteins, to obtain an accurate mass PINs the

preliminary analysis has been done using Maldi-TOF MS (detailed in Appendix III).

However, the results were inconclusive as sequences of the peptide were determined to

be unknown.

1 2 3 4 5

37kDa

15kDa

25kDa


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3.4 Results of antimicrobial activity tests

3.4.1 Antibacterial activity

The chloroplast-expressed purified PINs with different purification systems were tested

for antibacterial activity (as detailed in Chapter 2), using the protein stock (~1 mg/mL).

The synthetic peptide PuroA (FPVTWRWWKWWKG-NH2) was used as positive

control (Section 2.12.1). The negative control used was the TSP of N. benthamiana

infiltrated by 5’ and integrase modules that was purified based on affinity and

hydrophobic purifications. The activities were tested against Escherichia coli (ATCC

25922; a Gram negative bacterium) and Staphylococcus aureus (ATCC 25923; a Gram

positive bacterium); the strains of both can cause a range of illnesses in humans. The

minimum inhibitory concentration (MIC) was defined ‘the lowest concentration of an

antimicrobial agent which will inhibit the growth of a microorganism after overnight

incubation’ (Wiegand et al., 2008). The MIC results were observed by eye and by

measuring the optical density at 595 nm (OD595) of each well after 18 hours incubation

of cells with the peptide/protein. Activity of PuroA was consistent with previous work

in our laboratory (Phillips et al., 2011) (Table 3.4). The results confirmed in vitro

antibacterial abilities of plant-made recombinant PINs purified by His-tag purification

under native conditions and also by hydrophobic interaction purification. The PINs

purified under denaturing conditions had no observable activity. Both PINA and PINB

His-tag purified under native conditions showed activity against E. coli (MIC 64

μg/mL) which was less than that of the PuroA peptide (MIC 16 µg/µL) (Table 3.4;

Figure 3.27). PINA was observed to be active against S. aureus (MIC 64 μg/mL),

however, PINB showed higher activity (MIC 125 µg/mL). With hydrophobic

purification, both PINA and PINB showed better activity against E. coli (32 μg/mL),

and PINA showed the same activity (MIC 32 μg/mL) against S. aureus while PINB was

less active against it (64 μg/mL). These observations confirm the in vitro antimicrobial

abilities of recombinant PINs. Further, the PINs purified based on hydrophobic

interaction had better activity compared to both the His-tag purification systems. The

antibacterial activity of mixed PINA and PINB (1:1) (~1 mg/mL stock) were also tested

against both bacteria. The result observed same activities for both purification systems

(32 μg/mL and 64 μg/mL) (Table 3.4).


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Figure 3.27 Example of Minimum Inhibitory Concentration (MIC) assay for PuroA peptide and plant-made recombinant PIN proteins against E. coli. Peptide/protein dilution series from column 1 to 8; A: negative control (extract of leaf infiltrated with 5’+integrase modules only); B: positive control (PuroA peptide); C: rPINA (His-tag purification under denaturing conditions); D: rPINA (His-tag purification under native conditions); E: rPINA (hydrophobic purification); F: rPINB (His-tag purification under denaturing conditions); G: rPINB (His-tag purification under native conditions); H: rPINB (hydrophobic purification). Wells were inoculated with E. coli (ATCC 25922) and MIC recorded as the lowest concentration to completely inhibit growth. GC column= growth control of E. coli.

Table 3.4 Antibacterial activity of plant made recombinant PINA and PINB

Protein/peptide name Purification method

MIC (µg/mL)a

E. coli S. aureus PuroA

Positive control Synthetic peptide

FPVTWRWWKWWKG-NH2 16 (±0) 16 (±0)

rPINA His-tag purification under denaturing conditions >250 >250

rPINA His-tag purification under native conditions 64 (±0) 64 (±0)

rPINA Hydrophobic interaction purification 32 (±0) 32 (±0)

rPINB His-tag purification under denaturing conditions >250 >250

rPINB His-tag purification under native conditions 64 (±0) 125 (±0)

rPINB Hydrophobic interaction purification 32 (±0) 64 (±0)

PINA+PINB His-tag purification under native conditions 32 (±0) 64 (±0)

PINA+PINB Hydrophobic interaction purification 32 (±0) 64 (±0)

Negative controlb His-tag purification under native conditions >250 >250

Negative controlb Hydrophobic interaction purification >250 >250 aDefined as the lowest concentration that completely inhibited bacterial growth/ the mean of triplicate assays. bTSP of N. benthamiana infiltrated by 5’ and integrase modules only.

µµg/mL250 125 64 32 16 8 4 2 GC A

B

C

E

G

D

F

H


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3.4.2 Antifungal activity

The purified recombinant PINs were also tested for activity against five common phyto-

pathogenic fungi (Section 2.12.3). Rhizoctonia cerealis and Rhizoctonia solani are

responsible for sharp eye spot in wheat and sheath blight in rice, respectively (Hamada

et al., 2011; Taheri and Tarighi, 2011). Colletotrichum graminicola is responsible for

anthracnose in many cereals including wheat and maize (Dean et al., 2012). Drechslera

brizae is a fungus that causes post-harvest rot in crops (Naureen et al., 2009). Negative

control wells contained purified TSP of N. benthamiana infiltrated by 5’ and integrase

modules, and positive control wells contained PuroA. The MIC was defined as the

lowest concentration of the protein/peptide to inhibit fungal growth (Espinel-Ingroff and

Cantón, 2007), which was observed visually only. The results are presented in Table

3.5. The PIN proteins purified under both the native condition of His-tag purification

and the hydrophobic interaction purification showed activity against R. solani (250

µg/mL and 125 µg/mL) but were less active than the PuroA peptide (32 µg/mL) (Table

3.5; Figure 3.28). Proteins purified under denaturing conditions had no observable

antifungal activity.

Figure 3.28 Example of microdilution plate Minimum Inhibitory Concentration (MIC) assay for filamentous fungi against R. solani. Peptide/protein dilution series from column 1 to 9; A: negative control (extract of leaf infiltrated with 5’+integrase modules only); B: positive control (PuroA peptide); C: rPINA (His-tag purification under denaturing conditions); D: rPINA (His-tag purification under native conditions); E: rPINA (hydrophobic interaction purification); F: rPINB (His-tag purification under denaturing conditions); G: rPINB (His-tag purification under native conditions); H: rPINB (hydrophobic interaction purification). GC column=growth control of R. solani.


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Table 3.5 Antifungal activity of plant made recombinant PINA and PINB

aDefined as the lowest concentration of the peptide or protein to inhibit fungal growth/ the mean of triplicate assays. bTSP of N. benthamiana infiltrated by 5’ and integrase modules only.

Protein/ peptide name Purification method

MIC (µg/mL)a C.Gramini

cola D.brizae F. oxy-sporum

R. solani

R. cerealis

PuroA Positive control

Synthetic peptides FPVTWRWWKWWKG-NH

2 250 (±0) 250 (±0)

>500

32 (±0)

125 (±0)

PINA His-tag purification under denaturing conditions

>500 >500 >500 >500 >500

PINA His-tag purification under native conditions 500(±0) 500(±0) >500 250(±0) 250(±0)

PINA Hydrophobic interaction purification 500(±0) 500(±0) >500 125(±0) 250(±0)

PINB His-tag purification under denaturing conditions

>500 >500 >500 >500 >500

PINB His-tag purification under native conditions 500(±0) 500(±0) >500 250(±0) 250(±0)

PINB Hydrophobic interaction purification 500(±0) 500(±0) >500 125(±0) 125(±0)

PINA+PINB His-tag purification under native conditions 500(±0) 500(±0) >500 125(±0) 125(±0)

PINA+PINB Hydrophobic interaction purification 500(±0) 500(±0) >500 125(±0) 125(±0)

Negative controlb

His-tag purification under native conditions

>500 >500 >500 >500 >500

Negative controlb

Hydrophobic interaction purification

>500 >500 >500 >500 >500


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3.4.3 Protein stability at different temperature

The effect of temperature on the activity and stability of PIN proteins purified by His-

tag purification under native conditions were tested against E. coli and determined by

MIC assay. PIN proteins were stored at 37°C, RT (between 20°C-25°C), 4°C, -20°C

and -80°C for one week, then tested against E. coli. The results showed stable activity

against E. coli when stored at -20°C and -80°C. PIN proteins showed better activity

against E. coli at day one (MIC 64 μg/mL) compared to when stored at 4°C (MIC 250

μg/mL). The PuroA peptides retained with their original activity after one week of

incubation at room temperature (between 20°C-25°C), 4°C, -20°C and -80°C for one

week (Table 3.6).

Table 3.6 Stability of recombinant PINs at different incubation temperature

Peptide/Protein name

Incubation Temperature

MIC (µg/mL)a against E. coli at day 1

MIC (µg/mL)* against E. coli at day 7

PuroA Allb 16 (±0) 16 (±0) rPINA/rPINB -80°C 64 (±0) 64 (±0) rPINA/rPINB -20°C 64 (±0) 64 (±0) rPINA/rPINB 4°C 64 (±0) 250(±5) rPINA/rPINB 20°C-25°C 64 (±0) >250 rPINA/rPINB 37°C 64 (±0) >250

a The mean of triplicate assays, b PuroA not tested at 37°C.


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3.4.4 Haemolytic activity

The haemolytic activity of the purified PINs against sheep RBCs was determined as a

measure of toxicity to eukaryotic cells (Section 2.12.5). Positive haemolytic activity

was defined as the recombinant protein/peptide dose required to lyse 50% of the RBCs

(HD50). PBS (pH 7.4) and 1% Triton X-100 were included as a negative (zero%

haemolysis) and positive (100% haemolysis) controls, respectively. The recombinant

PINs purified with His-purification system under native condition at 125 μg/mL, 64

μg/mL, 32 μg/mL and 16 μg/mL did not show any notable activity (Figure 3.29, Table

3.7), confirming their non-lysis of mammalian cells and specificity against microbial

membranes. However, the PINs at 500 μg/mL and 250 μg/mL appeared to show higher

haemolytic activity than the peptide PuroA.

Figure 3.29 Haemolytic activity assays against sheep red blood cells (RBCs). Dilution series from column 1 to 6: 500 µg/mL, 250 µg/mL, 125 µg/mL, 64 µg/mL, 32 µg/mL and 16 µg/mL. rPINA: recombinant plant made PINA; rPINB: recombinant plant made PINB; Pos: Positive control 100% RBC lysis (1% Triton X-100); Neg: Negative control 0% RBC lysis (PBS, pH 7.4). Table 3.7 Haemolytic activity of plant-made recombinant PINs

Protein/Peptide

Percent haemolysis of sheep RBCs by recombinant PINs Haemolysis (HD50)a 500

(µg/mL) 250

(µg/mL) 125

(µg/mL) 64

(µg/mL) 32

(µg/mL) 16

(µg/mL) PuroA 15 10 6 4 0 0 >500 rPINA 32 22 10 6 0 0 >125 rPINB 20 20 8 6 0 0 >125

a Results represent the average of three separate experiments conducted in duplicate.


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3.5 Discussion

3.5.1 Expression

A number of studies with transgenic Pin genes have reported, the purpose being to test

their causative roles of PIN proteins in altering grain texture (Beecher et al., 2002a;

Hogg et al. 2004; Krishnamurthy and Giroux, 2001; Wada et al., 2010; Xia et al., 2008)

or testing whether they can impart in vivo antimicrobial activity (Faize et al., 2004; Kim

et al., 2012a; Krishnamurthy et al., 2001; Luo et al., 2008; Zhang et al., 2011) as

detailed in Chapter 1 (Sections 1.4.2 and 1.5.1). Further, mature full-length PINA and

PINB have been reported to be over-expressed in bacterial expression systems

(Capparelli et al., 2006; Capparelli et al., 2007) and in BY-2 Nicotiana tabacum cell

suspension culture (Sorrentino et al., 2009). In the BY-2 culture, only the recombinant

PINB targeted to the chloroplast was observed with an apparent size of 20 kDa and

yield of 0.35% of TSP but no signal were observed using two constructs for apoplast

targeting for both PINs (Sorrentino et al., 2009). Further, peptides similar to the PINs in

terms of being cysteine-rich and antimicrobial, but smaller, expressed in transgenic

tobacco lines represented only 0.1% of the TSP, and their expression either intra- or

extra-cellularly did not result in increased fungal resistance (De Bolle et al., 1996).

Plants as an alternative production platform for active recombinant therapeutic proteins

offers several advantages including lower production costs and high biomass volume.

In this study we used a transient transformation technology, the magnICON® for

production of PINs using N. benthamiana. This system is based on the deconstructed

tobacco mosaic virus (TMV) and is distinguished by the use of independent modules

harboring binary vectors, encoding different virus components, and allowing for DNA

delivery into plant cells by A. tumefaciens. The in vivo recombination, mediated by the

integrase (pICH14011) is accompanied with assembly of various pro-vectors, followed

by intron splicing, rendering an active viral replicon capable of producing the protein of

interest, and yields of up to 40% of TSP (Gleba et al. 2007). The replicon can move

systemically through the plant (Gleba et al. 2005; Marillonnet et al. 2004). The

assembly of recombinant proteins in plant tissues is critical for viability the plant

systems as bioreactors for therapeutic proteins (Streatfield 2007). The magnICON®

system is considered to be fast, requiring approximately 10 days for transformation and


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less than a month for the recovery of the expressed protein. This is highly desirable

compared to other systems such as chloroplast transformation which requires more than

two months, and nuclear stable transformation that requires several months (Patiño-

Rodríguez et al., 2013).

The successful expression of GFP proteins was visualized by examination under UV

light and in western blot using the anti-GFP antibody. Different constructs were used to

produce the PIN proteins in leaves of N. benthamiana. Both PIN proteins produced the

highest amount 10 days post infiltration when targeted to the chloroplast and cytosol.

As a commercial standard is not available for PINs, the 3% non-durum wheat in

Durotest kit for standard in ELISA assay was used to quantify PIN proteins; hence the

quantify estimates for PINs are approximate. The results demonstrate PINA and PINB

were transiently expressed in N. benthamiana and accumulated to ~0.83% and ~0.89%

of the TSP, respectively, following targeting to chloroplast. Western blot analysis by

Durotest antibody confirmed the size at approximately 45 kDa when the chloroplast and

cytosol targeting 5’ modules were used, and also with C-terminal SEKDEL added for

retention of the expressed protein in the ER. This finding suggests the formation of

aggregates not only inside chloroplasts but also in the cytosol and ER compartments of

the plant cell.

PINA is known to form aggregates in solutions with high ionic strength and low pH (Le

Bihan et al., 1996), correct folding being dependent on pH and a contributor to low

protein solubility (Issaly et al., 2001). Further, an earlier study has noted the importance

of protein aggregates of recombinant soybean agglutinin (rSBA) in N. benthamiana

necessary for its biological activity (Tremblay et al., 2011). Similar folding pattern has

also been observed, when proteins containing multiple disulphide bonds were expressed

in chloroplasts, including CTB-proinsulin (Ruhlman et al., 2007) and interferon-a2b

(Arlen et al., 2007) RC101 and PG1 (Lee et al., 2011b).


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3.5.2 Purification

Both PINA and PINB are disulphide-bonded proteins with unique TRD. PINs contain

10-Cys backbone proposed to form five disulphide bonds that have an important role in

protein stability and activity (detailed in Chapter 1). PINA and PINB have been

expressed in bacterial systems using His-tag (Capparelli et al., 2006) and GST-tag

(Capparelli et al., 2006; Capparelli et al., 2007). Furthermore, 3×FLAG N-terminal tag

was added for western blot analysis using mouse anti-flag monoclonal antibody which

detected recombinant PINB at ~20 kDa (Sorrentino et al., 2009).

Affinity tags can have role for promoting the solubility of the expressed fusion proteins

(Hearn and Acosta, 2001; Terpe, 2003; Waugh, 2005). Ideally, the tags should not

affect the structure and stability of the proteins. On the other hand, they may affect

proteins advantageously by increasing expression, solubility and stability (Arnau et al.,

2006; Hearn and Acosta 2001; Smyth et al., 2003; Terpe, 2003) and promote proper

folding (Smyth et al., 2003; Waugh, 2005). The His-tag with metal IMAC systems is

currently the most commonly used affinity tag (Listwan et al., 2004; Waugh, 2005).

Generally His-tag can interact with a chromatographic matrix like Ni-NTA resin as it is

most attracted to immobilized metal ions, such as cobalt, nickel and copper (Terpe,

2003; Waugh, 2005) the selectivity being based on the fact that the density of His

residues in this tag is higher than that on the surface of many proteins (Gaberc-Porekar

and Menart 2001) and other potential electron donor side chains include tryptophan and

cysteine (Scopes, 1993). Since both PINs contain tryptophan and cysteine, they may be

capable of binding to metal-chelate columns, hence both were fused translationally to a

small His-tag.

After affinity purification using the His-tag for PINs expressed in the chloroplast,

western blot analysis confirmed two bands at 45 kDa and 37 kDa. However the ~45

kDa proteins were observed as faint bands (Figure 3.20; lanes 6 and 7). In addition, for

the PINs protein sample infiltrated using the chloroplast 5’-module, the western blot

detected the small and faint band at ~30 kDa before purification (Figure 3.10 and 3.17;

lane 5). The expected sizes of the PIN monomers with His-tag were ~15 kDa. Thus,

the bands may correspond to multimers. Further, the His-tag could have affected the


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folding of the recombinant PIN proteins or incomplete cleavage of the chloroplast

transit peptide (Bruce, 2000; Emanuelsson et al., 1999), resulting in the size

discrepancy. The incomplete cleavage of the chloroplast transit peptide has also been

reported for the PyMSP4/5 antigen expressed in plants using the magnICON® system

chloroplast 5’-module (Webster et al., 2009). The artificial dicot chloroplast targeting

presequence for pICH12190 chlorolast targeted 5’ module coding for

[massmlssaavvatrasaaqasmvapftglksaasfpvtrkqnnlditsiasnggrvqca] (Marillonnet et al.,

2004; Dr. S. Marillonnet, personal commincation, April 2014) with estimated size of 6

kDa using the ‘compute pI/Mw tool’ at the ExPASy.

The hydrophobic purification system was attempted for C-terminally His-tagged PINs,

as this is widely used for the isolation of hydrophobic membrane proteins and

antimicrobial cationic peptides (Skosyrev et al., 2003). Hydrophobic patches of

proteins interact with a hydrophobic stationary phase. Ammonium sulfate decreases

solubility of most proteins, due to the salting-out effect, hence ethanol was used, being

less polar. The organic solvents, i.e., ethanol or n-butanol, enable protein precipitation

by electrostatic aggregations. Protein solubility during extraction is affected by a

complex mechanism using organic solvents. The salt promotes solubility of proteins by

preventing their electrostatic aggregation in the organic solvent (Samarkina et al., 2009).

However, the ethanol phase is also saturated with ammonium sulfate. It is important to

add ethanol as soon as possible after ammonium sulfate so as to avoid nonspecific

precipitation of target protein with other proteins. The two phases were formed within

three to five minutes after addition of ethanol and vigorous shaking, and centrifugation

was started as soon as the phases began to form. The n-butanol, a less polar and more

hydrophobic organic solvent than water was added to the ethanol phase to recover the

PINs in the lower aqueous phase. The hydrophobic interaction purification thus

allowed fast processing and ability to concentrate the sample. Butyl-Toyopearl column

was used for removing any compounds that were not visible in Coomassie Blue stained

gels. Both PINs were detected at ~37 kDa with the Durotest antibody, and this was the

only technique that showed the PINs with anti-His antibody in both multimer (~37 kDa)

and monomer (~15 kDa) bands. This finding is most significant and suggests that

hydrophobic purification affected the folding of PINs. Further, the results confirmed


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that the Durotest antibody is likely not able to detect monomeric forms of PINs.

However this antibody detected the positive at ~15 kDa which is protein complex of

friabilin with PINs as major part (detailed in section 1.2.1). Prior studies (Greenwell,

1992; Wiley et al., 2007) noted that the Durotest antibody probably did not react with

PINs after reduction of disulphide bonds or denaturation during fixation for

immunocytochemical analyses. The protein extracts from grain of a bread wheat line

and two transgenic lines of durum wheat expressing PINA and PINB were tested by

western blot using this antibody and unreduced PIN detected at 15 kDa (Wiley et al.,

2007). Recently, this antibody was used for immunolocalisation of PIN proteins in the

endosperm cell of transgenic rice (Fujiwara et al., 2014).

3.5.3 Characterisation of bioactivity

PINA and PINB have potent activities against a broad spectrum of microorganisms

(detailed in section 1.5). The recombinant PINA and PINB have been expressed in E.

coli and affinity-purified using His and GST tags, and tested against E. coli (MIC 30

μg/mL) and S. aureus (MIC 30 μg/mL) (Capparelli et al., 2006). The activity of

recombinant PINA expressed in E. coli and purified by affinity purification using NusA

fusion tag was reported to be active against E. coli (MIC 90 μg/mL) and S. aureus (MIC

150 μg/mL) (Miao et al., 2012). Furthermore, chloroplast targeted PINB in N. tabacum

cells has activity against E. coli (91% growth inhibition) (Sorrentino et al., 2009).

Several studies have revealed the successful production of low-cost and functional

antimicrobial peptides and proteins in transgenic tobacco (Jacob and Zasloff, 1994; Lee

et al., 2011b; Oey et al., 2009). Previously the ability of the Potato Virus X (PVX)

vector to produce an antimicrobial protein in N. benthamiana leaves has been reported

(Saioth et al. 2001). The human lactoferrin (hLfN) expressed in N. benthamiana using a

PVX is also active against E. coli and S. aureus (Li et al., 2004). The magnICON®

technology was also used in N. tabacum for producing the antimicrobial peptide

protegrin-1 (PG-1), which inhibited the growth of several bacterial and fungal

pathogens of humans (Patiño-Rodríguez et al., 2013). Hence the goal of this study was

to produce and purify functional PINs in planta.


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In the current study, purified soluble protein extracts from transiently-transformed N.

benthamiana leaves with magnICON® system, have been used for biological activity

assays of PIN proteins and sharp decrease observed in the viability of different

pathogens. Purified extracts of leaves infiltrated with PIN transgenes were added to in

vitro bioassays and showed successful expression of bioactive proteins. The

antibacterial activity of the His-tag purified PINA under native conditions showed a

similar activity against S. aureus and E. coli (MIC 64 μg/mL), however the purified

PINA using hydrophobic interaction system was more active against both S. aureus and

E. coli (MIC 32 μg/mL). His-tag purified PINB under native conditions had similar

activity as PINA against E. coli (MIC 64 μg/mL) but not against S. aureus (MIC 125

μg/mL). The activity results indicate that the hydrophobic interaction purified proteins

displayed 2-fold more activity against E. coli for both recombinant PINs. No inhibition

of microbial growth was detected with extracts from infiltrated leaves without PINs,

purified with both systems. The peptide PuroA (based on the TRD of wild type PINA)

used as positive control in the assays showed strong activity towards E. coli and S.

aureus (MIC 16 μg/mL), supporting previous reports (Jing et al., 2003; Phillips at al.,

2011). It is particularly noteworthy that the growth inhibition of both Gram-positive

and Gram-negative bacteria by recombinant PINs was high for both affinity and

hydrophobic systems, suggesting correct formation of disulphide bonds in the plant-

made PINs. The PIN proteins lose antimicrobial activity in the presence of 6 M urea.

Many proteins require a particular three-dimensional structure and disulphide bonds to

function. If the disulphide bonds are not formed correctly, the protein can degrade or

accumulate as insoluble inclusion bodies (Lesk, 2010). PINs, with their 10-Cys

backbone that forms five disulphide bonds may require these bonds for protein stability

and activity.

The antifungal activity of PIN proteins has been shown in vitro (Dubreil et al., 1998)

and in planta when Pin genes have been introduced transgenically, e.g., in rice

(Krishnamurthy et al., 2001), apple (Faize et al., 2004), durum wheat (Luo et al., 2008)

and wheat seeds overexpressing Pins (Kim et al., 2012a). This work further examined

the activity of the recombinant PINs against the five common fungi. Recombinant

plant-made PINs purified under His-tag and hydrophobic purification showed activity


- 124 -

against R. solani and R. cerealis. PINA and PINB, when purified by hydrophobic

interactions, showed better activity against most organisms tested, in comparison with

the His-tag purification under native conditions. A possible reason might be related to

the presence of more monomer forms of PINs in these preparations, or better protein

folding. Initially, the assay was carried out, which showed no activity against C.

graminicola, D. brizae and F. oxysporum at MIC 250 µg/mL. Hence, the assay was

repeated at a higher MIC (500 µg/mL) for both recombinant PINs. Activity was

observed only against C. graminicola and D. brizae (not for F. oxysporum) for PINA

and PINB purified with His-tag under native conditions and hydrophobic purification

systems. A possible explanation may be the stability of PINs and the time of incubation

(at 25°C in dark for 7 days). The results of stability testing indicate that the plant-made

PINs are not stable at 25°C or 37°C for 7 days. However, prior study in our laboratory

has noted the thermal stability of PuroA and PuroB peptides (Alfred et al., 2013a).

Previously in vitro studies indicated that the two proteins work synergistically with

enhanced activity against fungal pathogens (Capparelli et al., 2005). In this work when

PINA and PINB were mixed for the antimicrobial assay shown the inhibition against

bacteria and fungi and have shown synergy between the two proteins against fungi and

bacteria. The haemolytic activity assay displayed in PuroA at >500 µg/mL, however the

haemolytic activity of the recombinant PINs showed at >125 µg/mL. This variation

between PuroA and PIN proteins may be related to plant alkaloids in the preparations,

such as nicotine most abundant alkaloid in the genus Nicotiana and recently found to

exhibit haemolytic activity in vitro against human erythrocytes (Jasiewicz et al., 2014).

The findings in this chapter are highly promising in providing a methodology for

production of PINs as potential therapeutic agents against various mammalian and plant

pathogens as well as for use in hygiene and food industries. The two wild type PINs are

reported act in a co-operative manner when binding to the surface of starch granules.

The following chapter investigates PIN interaction in a plant system using the BiFC

system.

CHAPTER 4

Determination of the protein-protein interactions (PPI) of PIN proteins using Bimolecular Fluorescence

Complementation (BiFC) in planta

Chapter 4 Protein interaction

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4.1 Introduction

The wild type PIN proteins (PINA and PINB) are causatively associated with soft

wheat, while mutations in one or both Puroindoline genes result in hard textures, as

summarised in Chapter 1 (Sections 1.3 and 1.4). Both PINA and PINB associate with

biological membranes through their lipid-binding properties, which are also likely

involved in grain hardness determinants (Gautier et al., 1994; reviewed in Bhave and

Morris, 2008b; Morris, 2002; Nadolska-Orczyk et al., 2009). Transgenic studies (Hogg

et al., 2004; Martin et al., 2006; Swan et al., 2006, Wanjugi et al., 2007) have shown

that the PINs undergo some interaction or act in an inter-dependent manner in their

effects on grain texture. The two wild type PINs were found to act co-operatively to

prevent break-down of lipids in soft grains, mutations in either gene leading to higher

polar lipid degradation (Kim et al., 2012a). Furthermore, the tryptophan-rich domain

(TRD) has been considered the most important domain for the membrane-active nature

of PIN proteins. Despite the above literature on PINs, the nature of the interdependence

of the two proteins, interactions of PINs and any role of the TRD in these are unclear.

Addressing these important questions could help to improve wheat quality. Previous

work in our laboratory using the yeast 2-hybrid system provided evidence of in vivo

protein-protein interactions (PPI) in PIN proteins expressed in yeast cells (Ziemann et

al., 2008). The bimolecular fluorescence complementation (BiFC) assay is based on

complementation approaches to detect protein associations occurring in-vivo, and allows

visualisation of PPI in living cells by fluorescent signal (Gell et al., 2012; Hu et al.,

2002). This chapter investigates (i) whether any physical interactions occur in PINA

and PINB in planta using BiFC system; (ii) the effect of deletions of the hydrophobic

domain (HD) domains from both PINs and the TRD of PINA on any interactions, and

expression based on magnICON® system. The aims have been addressed using the

methodologies explained in section 2.14.


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4.2A Interaction studies for wild type PINA and PINB

4.2.1 Cloning of wild type PINA and PINB into pNBV vectors of the BiFC system

The ORF regions of the wild type alleles Pina-D1a (Genbank DQ363911), and Pinb-

D1a (Genbank DQ363913) encoding the mature PIN protein sections (lacking the N-

terminal signal peptide sequences) were amplified from the pICH-PINA-His and pICH-

PINB-His clones (detailed in Chapter 3). Gene-specific primers with the appropriate

restriction sites (Table 2.11) were used to introduce specific restriction sites at the 5’

and 3’ ends of the PCR fragments for cloning into the pNBV vectors (Section 2.14.2).

The start codon was added to all forward primers and the native stop codon was

removed for cloning into two pNBV vectors (gene-YN and gene-YC). The resulting

PCR fragments were 400 bp (Figure 4.1; lanes 1 and 2). These were double-digested

and cloned into four sets of double-digested pNBV vectors (gene-YN, gene-YC, YN-

gene and YC-gene), which allowed for fusions of each gene with the N-terminal 155

amino acids of YFP (the YN fragment) or the 84 amino acids of the C-terminal end of

YFP (the YC fragment). Empty pNBV vector DNAs were used to confirm the vector

size consistent with promoter, terminator and YFP fragments (~4 kb) (Figure 4.1; lanes

4 to 7). More details for pNBV vectors are shown in Figure 4.3.

Figure 4.1 Second-round PCR of gene sections encoding mature PINs and DNA plasmids of empty pNBV vectors. Lane M: DNA molecular weight marker, Hyperladder 1; lane 1: PINA (mature protein section of Pina-D1a with restriction sites); lane 2: PINB (mature protein section of Pinb-D1a with restriction sites); lane 3: negative control (no template); lane 4: DNA plasmid of pNBV vector (gene-YC); lane 5: DNA plasmid of pNBV vector (gene-YN); lane 6: DNA plasmid of pNBV vector (YN-gene); lane 7: DNA plasmid of pNBV (YC-gene).

M 1 2 3 4 5 6 7

4000bp

400bp

1000bp


- 128 -

The ligation mixtures of pNBV were transformed into E. coli Mach1 cells and grown on

LB plates with carbenicillin. Empty vectors were also transformed to be used as

controls. The presence of inserts was confirmed by colony-PCR (Figure 4.2) using

insert-specific primers (Table 2.11). Positive colonies (clones with inserts) were grown

in LB media containing carbenicillin. Plasmids were isolated from the positive clones

and used for PCR and double-digestion with restriction sites at the 5’ and 3’ ends of the

PINA and PINB fragments to confirm that the vectors contained the insert (data not

shown).

Figure 4.2 Colony PCR for preliminary selection of clones. Lane M: DNA molecular weight marker, Hyperladder 1; lane 1-3: pNBV-PINA product; lane 4 and 5: pNBV-PINB product; lane 6: negative control (no template). The above BiFC constructs were designated as pNBV-PINA-YN, pNBV-PINA-YC,

pNBV-YN-PINA and pNBV-YC-PINA for PINA and pNBV-PINB-YN, pNBV-PINB-

YC, pNBV-YN-PINB and pNBV-YC-PINB for PINB (Figure 4.3). They contain the

35S promoter of the cauliflower mosaic virus (862 bp), terminator of the Nos gene

(NosT: 264 bp), lacZ gene (215 bp), a linker between YN or YC and the gene (54 bp),

and yellow fluorescent protein (YFP) (YN=N-terminal fragment, amino acids 1 to 155

(465 bp), or YC=C-terminal fragment, amino acids 156 to 238 (249 bp)). The BiFC

cassettes in pNBV were released from pNBV by NotI digestion and inserted into the

plant expression vector (pMLBART; Figure 2.10). The full sequences of pNBV

cassettes without PINs are shown in Appendix IV.

M 1 2 3 4 5 6

600bp

400bp

1000bp


- 129 -

Figure 4.3 The pNBV module constructs encoding mature PINA and PINB proteins. 35S: promoter of the cauliflower mosaic virus; NosT: terminator of the Nos gene; YFP: yellow fluorescent protein (YN=N-terminal fragment 1 to 155 amino acid/YC=C-terminal fragment 156 to 238 amino acid). A1: pNBV-PINA-YN; A2: pNBV-PINA-YC; A3: pNBV-YN-PINA; A4: pNBV-YC-PINA; B1: pNBV-PINB-YN; B2: pNBV-PINB-YC; B3: pNBV-YN-PINB; B4: pNBV-YC-PINB.

LacZ PINA 35S NosT YFP-(YN)

NotI NotI XbaI XmaI

NosT 35S LacZ YFP-(YC)

NotI NotI XbaI XmaI

PINA

35S LacZ YFP-(YN)

NotI NotI

Linker

BamHI EcoRI

NosT PINA

35S LacZ YFP-(YC)

NotI NotI

Linker

BamHI EcoRI

NosT PINA

LacZ 35S NosT YFP-(YN)

NotI NotI XbaI XmaI

PINB


NotI NotI XbaI XmaI

PINB

35S LacZ YFP-(YN)

NotI NotI

Linker

BamHI EcoRI

NosT PINB

35S LacZ YFP-(YC)

NotI NotI

Linker

BamHI EcoRI

NosT PINB

A1

A2

A3

A4

B2

B1

B4

B3


- 130 -

The constructs were transformed into E. coli strain cells and screened by colony PCR

with insert-specific primers (Table 2.11) followed by DNA sequencing. Alignment

with the reference sequences; Pina-D1a (DQ363911) (Figure 4.4) and Pinb-D1a

(DQ363913) (Figure 4.5) showed 100% identity.

Figure 4.4 Alignments sequence of PINA in pNBV construct. A: DNA sequence of mature putative Pina-D1a genes cloned in the pNBV (gene-YN) and pNBV (gene-YC) vectors. B: DNA sequence of mature putative Pina-D1a genes cloned in the pNBV (YN-gene) and pNBV (YC-gene) vectors. In both A and B, the line 1 is sequence of the reported WT Pina-D1a gene (DQ363911).

10 20 30 40 50 60. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G A T G T T G C T G G C G G G G G T G G T G C T C A A C A A T G C C C T G T A G A G A C ApNBV-PINA-YN G G G G A C T C T A G A A T G G A T G T T G C T G G C G G G G G T G G T G C T C A A C A A T G C C C T G T A G A G A C ApNBV-PINA-YC ~ ~ ~ G A C T C T A G A A T G G A T G T T G C T G G C G G G G G T G G T G C T C A A C A A T G C C C T G T A G A G A C A

70 80 90 100 110 120. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature A A G C T A A A T T C A T G C A G G A A T T A C C T G C T A G A T C G A T G C T C A A C G A T G A A G G A T T T C C C GpNBV-PINA-YN A A G C T A A A T T C A T G C A G G A A T T A C C T G C T A G A T C G A T G C T C A A C G A T G A A G G A T T T C C C GpNBV-PINA-YC A A G C T A A A T T C A T G C A G G A A T T A C C T G C T A G A T C G A T G C T C A A C G A T G A A G G A T T T C C C G

130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature G T C A C C T G G C G T T G G T G G A A A T G G T G G A A G G G A G G T T G T C A A G A G C T C C T T G G G G A G T G TpNBV-PINA-YN G T C A C C T G G C G T T G G T G G A A A T G G T G G A A G G G A G G T T G T C A A G A G C T C C T T G G G G A G T G TpNBV-PINA-YC G T C A C C T G G C G T T G G T G G A A A T G G T G G A A G G G A G G T T G T C A A G A G C T C C T T G G G G A G T G T

190 200 210 220 230 240. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature T G C A G T C G G C T C G G C C A A A T G C C A C C G C A A T G C C G C T G C A A C A T C A T C C A G G G G T C A A T CpNBV-PINA-YN T G C A G T C G G C T C G G C C A A A T G C C A C C G C A A T G C C G C T G C A A C A T C A T C C A G G G G T C A A T CpNBV-PINA-YC T G C A G T C G G C T C G G C C A A A T G C C A C C G C A A T G C C G C T G C A A C A T C A T C C A G G G G T C A A T C

250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature C A A G G C G A T C T C G G T G G C A T C T T C G G A T T T C A G C G T G A T C G G G C A A G C A A A G T G A T A C A ApNBV-PINA-YN C A A G G C G A T C T C G G T G G C A T C T T C G G A T T T C A G C G T G A T C G G G C A A G C A A A G T G A T A C A ApNBV-PINA-YC C A A G G C G A T C T C G G T G G C A T C T T C G G A T T T C A G C G T G A T C G G G C A A G C A A A G T G A T A C A A

310 320 330 340 350 360. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature G A A G C C A A G A A C C T G C C G C C C A G G T G C A A C C A G G G C C C T C C C T G C A A C A T C C C C G G C A C TpNBV-PINA-YN G A A G C C A A G A A C C T G C C G C C C A G G T G C A A C C A G G G C C C T C C C T G C A A C A T C C C C G G C A C TpNBV-PINA-YC G A A G C C A A G A A C C T G C C G C C C A G G T G C A A C C A G G G C C C T C C C T G C A A C A T C C C C G G C A C T

370 380 390. . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature A T T G G C T A T T A C T G G T G A pNBV-PINA-YN A T T G G C T A T T A C T G G C C C G G G A T G G A G C A ApNBV-PINA-YC A T T G G C T A T T A C T G G C C C G G G A T G T A C

A

B


- 131 -

Figure 4.5 Alignments sequence of PINB in pNBV construct. A: DNA sequence of mature putative Pinb-D1a genes cloned in the pNBV (gene-YN) and pNBV (gene-YC) vectors. B: DNA sequence of mature putative Pinb-D1a genes cloned in the pNBV (YN-gene) and pNBV (YC-gene) vectors. In both A and B, the line 1 is sequence of the reported WT Pinb-D1a gene (DQ363913).

One each of the eight pMLBART constructs was individually transformed into the

Agrobacterium strain GV3101. Screening of the clones containing inserts was carried

out by colony-PCR using primer listed in Table 2.11 (data not shown).

10 20 30 40 50 60. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G A A G T T G G C G G A G G A G G T G G T T C T C A A C A A T G T C C G C A G G A G C G GpNBV-PINB-YN ~ ~ ~ G A C T C T A G A A T G G A A G T T G G C G G A G G A G G T G G T T C T C A A C A A T G T C C G C A G G A G C G GpNBV-PINB-YC G G G G A C T C T A G A A T G G A A G T T G G C G G A G G A G G T G G T T C T C A A C A A T G T C C G C A G G A G C G G

70 80 90 100 110 120. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature C C G A A G C T A A G C T C T T G C A A G G A T T A C G T G A T G G A G C G A T G T T T C A C A A T G A A G G A T T T TpNBV-PINB-YN C C G A A G C T A A G C T C T T G C A A G G A T T A C G T G A T G G A G C G A T G T T T C A C A A T G A A G G A T T T TpNBV-PINB-YC C C G A A G C T A A G C T C T T G C A A G G A T T A C G T G A T G G A G C G A T G T T T C A C A A T G A A G G A T T T T

130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature C C A G T C A C C T G G C C C A C A A A A T G G T G G A A G G G C G G C T G T G A G C A T G A G G T T C G G G A G A A GpNBV-PINB-YN C C A G T C A C C T G G C C C A C A A A A T G G T G G A A G G G C G G C T G T G A G C A T G A G G T T C G G G A G A A GpNBV-PINB-YC C C A G T C A C C T G G C C C A C A A A A T G G T G G A A G G G C G G C T G T G A G C A T G A G G T T C G G G A G A A G

190 200 210 220 230 240. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature T G C T G C A A G C A G C T G A G C C A G A T A G C A C C A C A A T G T C G C T G T G A T T C T A T C C G G C G A G T GpNBV-PINB-YN T G C T G C A A G C A G C T G A G C C A G A T A G C A C C A C A A T G T C G C T G T G A T T C T A T C C G G C G A G T GpNBV-PINB-YC T G C T G C A A G C A G C T G A G C C A G A T A G C A C C A C A A T G T C G C T G T G A T T C T A T C C G G C G A G T G

250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature A T C C A A G G C A G G C T C G G T G G C T T C T T G G G C A T T T G G C G A G G T G A G G T A T T C A A A C A A C T TpNBV-PINB-YN A T C C A A G G C A G G C T C G G T G G C T T C T T G G G C A T T T G G C G A G G T G A G G T A T T C A A A C A A C T TpNBV-PINB-YC A T C C A A G G C A G G C T C G G T G G C T T C T T G G G C A T T T G G C G A G G T G A G G T A T T C A A A C A A C T T

310 320 330 340 350 360. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature C A G A G G G C C C A G A G C C T C C C C T C A A A G T G C A A C A T G G G C G C C G A C T G C A A G T T C C C T A G TpNBV-PINB-YN C A G A G G G C C C A G A G C C T C C C C T C A A A G T G C A A C A T G G G C G C C G A C T G C A A G T T C C C T A G TpNBV-PINB-YC C A G A G G G C C C A G A G C C T C C C C T C A A A G T G C A A C A T G G G C G C C G A C T G C A A G T T C C C T A G T

370 380. . . . | . . . . | . . . . | . . . . | . . . .

PINB-Mature G G C T A T T A C T G G T G A pNBV-PINB-YN G G C T A T T A C T G G C C C G G G A T G G A GpNBV-PINB-YC G G C T A T T A C T G G C C C G G G A T G

A

B


- 132 -

4.2.2 BiFC system for transient expression in N. bethamiana leaves

Agrobacterium harbouring BiFC constructs were infiltrated into eight week old N.

benthamiana leaves. The fluorescence started to appear 3-4 days post infiltration (dpi).

In general, the best observation time for BiFC fluorescence was 3 dpi, after which the

signal began to fade. The expression of the fusion proteins was observed using a highly

active promoter i.e., the CaMV 35S promoter. In order to ensure that the fluorescence

resulted from the specific PIN protein-protein interactions and not from non-specific

binding of the two YFP fragments, or from background or autofluorescence,

infiltrations with buffer only, or with just one PIN-YFP construct, or YN and YC

construct without PIN ligation, were used as negative controls. Any fluorescence

obtained with such constructs would reflect non-specific interactions of the expressed

proteins. In addition, non-treated leaves were used to detect background fluorescence.

4.2.3 Post-transcriptional gene silencing (PTGS) in transient system

N. benthamiana plants were transiently co-transformed with the desired construct and

p19. The p19 protein from tomato bushy stunt virus (TBSV) (Section 2.14.7), by co-

expression of a viral-encoded suppressor of gene silencing can prevent post-

transcriptional gene silencing (PTGS) in a transient assay system (Voinnet et al., 2003).

This strategy has been used in a number of transient assays to enhance transgene

expression (Grefen et al., 2008; Hellens et al., 2005; Wroblewski et al., 2005), including

BiFC analyses (Schütze et al., 2009; Walter et al., 2004; Waadt et al., 2008). To

determine if co-expression of BiFC constructs and the p19 could enhance PIN

expressions, an Agrobacterium LB4404 containing the p19 was co-infiltrated with BiFC

constructs into N. benthamiana leaves. This led to a strong increase in fluorescence

signal, as observed in individual cells. This improvement in signal and experimental

reliability was the reason for the p19 to be included in the subsequent transient BiFC

analyses.


- 133 -

4.2.4 Fluorescence detection for the wild type PIN protein-protein interactions

The infiltration experiments were carried out in at least three replicates. The lower

epidermal layers of infiltrated leaves were examined by fluorescence microscopy 3 dpi.

No fluorescence signal was detected in non-treated leaves and in leaves infiltrated with

negative controls (Figure 4.6A and 4.6B). Background fluorescence was often observed

in cells surrounding wounded tissues of leaves, probably caused by the infiltration

procedure. These areas became yellowish 3 dpi.

A B

Figure 4.6 BiFC visualisation in N. benthamiana leaves. A and B: negative control (YC+YN without PINs) (20×magnification) and (200×magnification) respectively. Leaves co-infiltrated with a 1:1 volume ratio of Agrobacteria harbouring PINA-YC and

YN-PINB constructs showed fluorescence, which was stronger than the background

(Figure 4.7A-D) thus indicating an interaction between the fusion proteins.

Remarkably, the strong fluorescence was only observed in the PINA-YC+YN-PINB. In

contrast, leaves infiltrated with combinations of other PIN-BiFC constructs led to a

weak fluorescence in a small number of leaf cells, lack of fluorescence. This finding

suggests that the orientation of the PIN proteins is important for the interactions.


- 134 -

A B

C D

Figure 4.7 BiFC visualisation of PINA-PINB interactions in N. benthamiana leaves. A and B: interaction of PINA-PINB, in direction of PINA-YC+YN-PINB (20×magnification); C and D: interaction of PINA-PINB, in direction of PINA-YC+YN-PINB (200×magnification).

Since the structural basis of complex formations for PIN proteins is not clear, the two

interacting proteins were fused N- and C-terminally to the C- and N- terminal part of

YFP. Furthermore, potential homodimerisation was also investigated. Notably, the

fluorescence signals were observed in YN-PINB and YC-PINB constructs (Figure 4.8A

and 4.8B). However, no fluorescence was detected for homodimerisation in wild type

of PINA (Figure 4.8C and 4.8D) tested in different orientations (Table 4.1). From

images of fluorescence microscopy it seemed that PINA and PINB most likely

presented interactions in the cytoplasm. The small fluorescence dots illustrated likely

location in the chloroplast, and the small rings as Golgi compartments, for PINA-PINB

(Figure 4.7A-D) and especially for PINB-PINB (Figure 4.8A and B) interactions.


- 135 -

A B

C D

Figure 4.8 BiFC visualisation of PINA-PINA and PINB-PINB interactions in N. benthamiana leaves. A: interaction of PINB-PINB, in direction of YN-PINB+YC-PINB (20× magnification); B: interaction of PINB-PINB, in direction of YN-PINB+YC-PINB (200× magnification); C: interaction of PINA-PINA (20×magnification); D: interaction of PINA-PINA (200× magnification).

4.2.5 Western blot analysis of wild type PIN protein-protein interactions

Western blot analysis using the Durotest antibody was used to verify the co-expression

of the interacting forms of PINs. The leaves were harvested after 3 dpi and used for

microscopy. The total soluble protein (TSP) was extracted from leaves with the

fluorescence signal. The amounts of TSP were quantified using Bradford assay and

about 40 µg of TSP were loaded on 15% gel for further analysis by western blot. One

intense band at approximately 45 kDa was detected by western blot from the TSP of

infiltrated leaf with the PINA-YC+YN-PINB constructs (Figure 4.9; lanes 4 and 5).


- 136 -

The western blot also detected YN-PINB+YC-PINB interacting forms (Figure 4.9; lane

2); however the band size appeared to be slightly higher than PINA+PINB interactions.

Figure 4.9 Determination of interacting form of PINA+PINB and PINB+PINB by western blot analysis from TSP extract. Lane 1: YFP construct without PIN (negative control); lane 2: interacting form of recombinant PINB and PINB (YN-PINB+YC-PINB); lane 3: void lane (no sample loaded); lanes 4 and 5: interacting form of recombinant PINA and PINB (PINA-YC+YN-PINB); lane 6: 3% non-durum wheat as positive for antibody.

4.2.6 Gel detection of YFP fluorescence for wild type PIN protein-protein

interactions

In order to confirm the protein complexes, native electrophoresis was used on the same

samples as those used for western blot, for subsequent imaging of in-gel YFP

fluorescent under UV light. Fluorescent bands were detected for PINA-YC+YN-PINB

and for YN-PINB+YC-PINB interactions (Figure 4.10; lanes 1 and 2) which was also

confirming the above results of fluorescent microscopy and western blot. No bands

were detected for the negative controls (lanes 3 and 4).

1 2 3 4 5 6

50kDa

37kDa

75kDa

15kDa


- 137 -

Figure 4.10 Native gel electrophoresis and UV imaging of YFP fluorescence of PIN protein complexes. Lane 1: interacting form of recombinant PINA and PINB (PINA-YC+YN-PINB); lane 2: interacting form of recombinant PINB and PINB (YN-PINB+YC-PINB); lanes 3 and 4: YFP construct without PIN (negative).

4.2B Interaction studies of mutagenised PINA and PINB

4.2.7 Cloning of the previously made constructs PINA∆HD and PINB∆HD into

pNBV vectors

Site-Directed Mutagenesis (SDM) PCR had been earlier carried out by Dr R. Alfred

(Swinburne University of Technology) to delete the predicted hydrophobic domain

(HD) at positions 75-85 in the putative mature PINA (PINA∆HD) and at positions 75-

83 in the putative mature PINB (PINB∆HD) in yeast 2-hybrid system vectors pGBK

(details in section 2.15.1). These clones kindly provided by Dr R. Alfred, were used in

the present work as templates for PCR (Figure 4.11) to clone into pNBV vectors, so as

to see whether these deletions significantly affect the likely PPI of PINs. The primers

PIN-Forward and PIN-Reverse (Section 2.14.3) were used to introduce specific

restriction sites (XbaI and XmaI for PINA∆HD and EcoRI and BamHI for PINB∆HD)

at the 5’ and 3’ ends of the PCR fragments. PCR products of ~400 bp were obtained for

PINA∆HD and PINB∆HD (Figure 4.11), consistent with sizes of the gene sections

encoding putative mature PINA without HD (330 bp) or PINB without HD (333 bp) and

the added specific restriction sites (12 bp).

1 2 3 4


- 138 -

Figure 4.11 Amplification of inserts from PINA∆HD and PINB∆HD constructs. PCR products from clones in pGBK vectors using insert-specific primers. Lane 1: DNA molecular weight marker, Hyperladder l; lanes 2 and 3: PINA∆HD; lanes 4 and 5: PINB∆HD.

The PCR fragments were digested at restriction sites and cloned into cloning site of

pNBV vector to allow for translational fusions with the YN or YC fragments of YFP, as

above. The BiFC constructs were designated: pNBV-PINA∆HD-YC, pNBV-YN-

PINB∆HD and pNBV-YC-PINB∆HD (Figure 4.12). The cassettes in pNBV were

released from pNBV by NotI digestion, inserted into pMLBART transformed into E.

coli cells and screened by colony PCR with insert-specific primers (Table 2.11)

followed by DNA sequencing. Alignment with the reference sequences; Pina-D1a

(DQ363911) and Pinb-D1a (DQ363913) that showed 100% identity with the

corresponding sections (Figures 4.13 and 4.14).


- 139 -

Figure 4.12 The pNBV module constructs generated encoding mutant PIN proteins. 35S: promoter of the cauliflower mosaic virus; NosT: terminator of the Nos gene; YFP: yellow fluorescent protein (YN=N-terminal fragment 1 to 155 amino acid/YC=C-terminal fragment 156 to 238 amino acid). A: pNBV-PINA∆HD-YC; B: pNBV-YN-PINB∆HD; C: pNBV-YC-PINB∆HD.

Figure 4.13 Sequences of PINA∆HD in pNBV construct. DNA sequences of the mutated Pina-D1a genes cloned in pNBV vector. The top line (PINA) is the deduced protein sequence of the reported WT Pina-D1a (Genbank ABD72477, mature protein), line 2 is the DNA sequence of the reported Pina-D1a gene (DQ363911) and line 3 is the DNA sequence of PINA∆HD in pNBV (gene-YC) vector. The HD (IQGDLGGIFGF) is underlined.


NotI NotI XbaI XmaI

PINA∆HD

35S LacZ YFP-(YN)

NotI NotI

Linker

BamHI EcoRI

NosT PINB∆HD

35S LacZ YFP-(YC)

NotI NotI

Linker

BamHI EcoRI

NosT PINB∆HD

A

B

C


- 140 -

Figure 4.14 Sequences of PINB∆HD in pNBV construct. DNA sequences of the mutated Pinb-D1a genes cloned in pNBV vector. The top line (PINB) is the deduced protein sequence of the reported WT Pinb-D1a (Genbank ABD72479, mature protein), line 2 is the DNA sequence of the reported Pinb-D1a gene (DQ363913), line 3 is the DNA sequence of PINB∆HD in pNBV-YN-gene vector, line 4 is the DNA sequence of PINB∆HD in pNBV-YC-gene. The HD (VIQGRLGGF) is underlined. Each pMLBART construct was transformed into the Agrobacterium strain GV3101.

Screening of the clones containing inserts was confirmed by antibiotic (spectinomycin

plus rifampicin) selection and colony PCR using primer listed in Table 2.11 (data not

shown).


- 141 -

4.2.8 Detection of mutant PINA∆HD and PINB∆HD protein-protein interactions

by fluorescence and western blot

The potential role of the hydrophobic domain (HD) in any PPI of PINA and PINB was

investigated through infiltration of the above constructs into N. benthamiana leaves side

by side on the same leaf (Figure 4.15), with the wild type PIN construct which had

shown the fluorescent signal for interactions (Figure 4.7). Since the PINs had shown

fluorescent signal in one orientation (PINA-YC+YN-PINB), the mutant PINs were

tested as same orientation, i.e., PINA∆HD-YC+YN-PINB∆HD. However the results

showed no fluorescence. The interaction between PINA∆HD and wild type PINB

(PINA∆HD-YC+YN-PINB) and also wild type PINA and PINB∆HD (PINA-YC+YN-

PINB∆HD) led to no fluorescence.

Figure 4.15 Infiltrated mixtures of constructs into N. benthamiana leaves side by side for detect and compare the interaction of wild type and mutant form of PIN proteins.

Further, TSP were extracted from infiltrated areas, quantified using Bradford assay and

about 40 µg of TSP electrophoresed. Western blot analysis resulted in very faint bands

at ~45 kDa (Figure 4.16; lanes 2, 3, 4) compared to the wild type controls (lanes 5, 7).

The results suggest that the HD deletion in the direction led to significant reduction in

interactions of PINA∆HD-YC with YN-PINB∆HD and also of the self-interacting

PINB (YN-PINB∆HD+YC-PINB∆HD) (Figure 4.16; lane 4).

Infiltrated area with PINA-YC

+ YN-PINB

Infiltrated area with PINA∆HD-YC

+ YN-PINB∆HD


- 142 -

Figure 4.16 Western blot analysis to determine the interacting forms of wild type and mutant PIN proteins. Lane 1: YFP construct without insert (negative control); lanes 2 and 3: interacting form of recombinant PINA∆HD-YC+YN-PINB∆HD; lane 4: interacting form of recombinant YN-PINB∆HD+YC-PINB∆HD; lane 5 and 6: interacting form of recombinant PINA-YC+YN-PINB; lane 7: interacting form of recombinant YN-PINB+YC-PINB.

4.2.9 Cloning of PINA∆TRD into pNBV vectors

PCR was also conducted by Dr R. Alfred to delete the TRD at positions 34-46 in the

mature PINA protein (PINA∆TRD) in the yeast 2-hybrid system vectors pGBK (details

in section 2.15.1). The clone (kindly donated by Dr R. Alfred) was also used in the

present work as template for PCR (data not shown) to clone in pNBV. The primers

PIN-Forward and PIN-Reverse (Section 2.14.3) were used to insert restriction sites

(XbaI and XmaI) at the 5’ and 3’ ends of the PCR fragment. The resulting PCR

fragments were ~400 bp for PINA∆TRD was consistent with the gene sections encoding

putative mature PINA without the TRD (324 bp). The PCR fragments were digested

and cloned into pNBV as above to obtain the construct pNBV-PINA∆TRD-YC with the

translational fusion with the YC fragment of YFP (Figure 4.17). The BiFC cassette in

pNBV was released by NotI digestion, inserted into pMLBART transformed into E. coli

which cells and screened by colony PCR with insert-specific primers (Table 2.11),

followed by DNA sequencing. Alignment with Pina-D1a (DQ363911) showed 100%

identity (Figure 4.18). The pMLBART construct was then transformed into the

Agrobacterium strain GV3101.

1 2 3 4 5 6 7

50kDa

75kDa

37kDa

15kDa


- 143 -

Figure 4.17 The pNBV module construct generated encoding PINA∆TRD protein. 35S: promoter of the cauliflower mosaic virus; NosT: terminator of the Nos gene; YFP: yellow fluorescent protein (YC=C-terminal fragment 156 to 238 amino acid); pNBV-PINA∆TRD-YC.

Figure 4.18 Sequences of PINA∆TRD in pNBV construct. DNA sequences of the mutated Pina-D1a genes cloned in pNBV vector. The top line (PINA) is the deduced protein sequence of the reported WT Pina-D1a (Genbank ABD72477, mature protein), line 2 is the DNA sequence of the reported Pina-D1a gene (DQ363911), and line 3 is the DNA sequence of PINA∆TRD in pNBV (gene-YC) vector. The TRD (FPVTWRWWKWWKG) is underlined.


NotI NotI XbaI XmaI

PINA∆TRD


- 144 -

4.2.10 Fluorescence detection for mutant PINA∆TRD protein-protein interactions

Infiltration of the relevant constructs into N. benthamiana leaves showed no

fluorescence or only background fluorescence for PINA∆TRD-YC+YN-PINB. Further,

no fluorescence signal was detected for interaction between PINA∆TRD-YC+YN-

PINB∆HD. These results suggest that the TRD deletion in PINA leads to significant

change in the interactions of PIN. The full set of PIN interactions tested by the BiFC

assay is shown in Table 4.1.

Table 4.1 BiFC constructs for PIN proteins (wild type and mutant) to detect fluorescence signal

PINA∆TRD=Deletion of the tryptophan-rich domain (TRD), location at 34-46 amino acid in PINA PINA∆HD=Deletion of a hydrophobic domain (HD), location at 75-85 amino acid in PINA PINB∆HD=Deletion of a hydrophobic domain (HD), location at 75-85 amino acid in PINA

No Construct 1 Construct 2 Heterodimerisation

wild type PIN proteins Fluorescence

signal 1 PINA-YC YN-PINB PINA+PINB Yes 2 PINA-YC PINB-YN PINA+PINB No 3 PINA-YN YC-PINB PINA+PINB No 4 PINA-YN PINB-YC PINA+PINB No 5 YC-PINA PINB-YN PINA+PINB No 6 YC-PINA YN-PINB PINA+PINB No 7 YN-PINA PINB-YC PINA+PINB No 8 YN-PINA YC-PINB PINA+PINB No

No Construct 1 Construct 2 Homodimerisation

wild type PINA proteins Fluorescence

signal 1 YN-PINA PINA-YC PINA+PINA No 2 YN-PINA YC-PINA PINA+PINA No 3 PINA-YN YC-PINA PINA+PINA No 4 PINA-YN PINA-YC PINA+PINA No


wild type PINB proteins Fluorescence

signal 1 YN-PINB PINB-YC PINB+PINB No 2 YN-PINB YC-PINB PINB+PINB Yes 3 PINB-YN YC-PINB PINB+PINB No 4 PINB-YN PINB-YC PINB+PINB No

No Construct 1 Construct 2 Heterodimerisation

of mutant PIN proteins Fluorescence

signal 1 PINA∆HD-YC YN-PINB∆HD PINA∆HD+PINB∆HD No 2 PINA∆HD-YC YN-PINB PINA∆HD+PINB No 3 PINA-YC YN-PINB∆HD PINA+PINB∆HD No 4 PINA∆TRD-YC YN-PINB∆HD PINA∆TRD+PINB∆HD No 5 PINA∆TRD-YC YN-PINB PINA∆TRD+PINB No


of mutant PINB proteins Fluorescence

signal 1 YN-PINB YC-PINB∆HD PINB+PINB∆HD No 2 YN-PINB∆HD YC-PINB∆HD PINB∆HD+PINB∆HD No


- 145 -

4.2.11 Expression of mutant PIN proteins in magnICON® system

To determine the roles of the HD in both PINA and PINB and the TRD in PINA on

expression of PIN proteins in planta, the magnICON® system was also used. The 3’

modules pICH11599 containing PINA∆HD, PINB∆HD and PINA∆TRD were

constructed as detailed in Chapter 2 (Figure 4.19). This was followed by transformation

of the plasmids into E. coli Mach1 cells and purification of plasmids. Sequencing

confirmed the correct deletions and 100% identity of the rest of the sequence with the

wild type alleles (Figures 4.20 and 4.21).

Figure 4.19 The 3’ module constructs encoding mutant PIN proteins with His-tag. A: pICH-PINA∆TRD-His; B: pICH-PINA∆HD-His C: pICH-PINB∆HD-His


RB LB EcoRI BamHI

PINA∆TRD


RB LB EcoRI BamHI

PINA∆HD

His PINB∆HD Tnos NPT Pnos Intron 3’NTR Tnos AttB

RB LB NcoI SacI

A

B

C


- 146 -

Figure 4.20 Sequences of PINA∆HD-His and PINA∆TRD-His in the 3’ module pICH11599. The top line (PINA) is the deduced protein sequence of the reported WT Pina-D1a (Genbank ABD72477, mature protein), line 2 is the DNA sequence of the reported Pina-D1a gene (DQ363911), line 3 is the DNA sequence of PINA∆HD-His, line 4 is the DNA sequence of PINA∆TRD-His. The HD (VIQGRLGGF) and TRD (FPVTWRWWKWWKG) are underlined. His-tag sequences are in box.


- 147 -

Figure 4.21 Sequences of PINB∆HD-His in 3’ module pICH11599. The top line (PINB) is the deduced protein sequence of the reported WT Pinb-D1a (Genbank ABD72479, mature protein), line 2 is the DNA sequence of the reported Pinb-D1a gene (DQ363913), line 3 is the DNA sequence of PINB∆HD-His. The HD (VIQGRLGGF) is underlined. His-tag sequence is in box. The plasmids were electroporated into Agrobacterium GV3101 and infiltrated in N.

benthamiana leaves side by side with wild type of PINA and PINB. Approximately

~40 µg of TSP was loaded in each lane, followed by western blot. Interestingly, bands

were detected at ~45 kDa for expressions of chloroplast targeted PINA∆HD and

PINB∆HD, suggesting aggregate forms (Figure 4.22; lanes 1 and 2) similar to wild

type PINA and PINB (lanes 3 and 4), but PINA∆TRD was not detected (lane 5). The

results suggest that PINA with deletion of TRD may be degraded, or the Durotest

antibody was not able to detect it. The cytosol targeted PINs indicated the same result

(data not shown).


- 148 -

Figure 4.22 Determination of wild type and mutant PIN proteins expressions in infiltrated leaves with chloroplast viral vector module by western blotting of TSP extract. Lane1: chloroplast targeted PINA∆HD; lane 2: chloroplast targeted PINB∆HD; lane 3: chloroplast targeted PINA; lane 4: chloroplast targeted PINB; lane 5: chloroplast targeted PINA∆TRD; lane 6: negative control (5’+integrase modules only).

1 2 3 4 5 6

50kDa

37kDa

15kDa


- 149 -

4.2.12 Bioinformatics analysis of PIN sequences for potential interaction domain(s)

In order to gain more insights into any particular residues/domains that could be

involved in the interaction of PIN proteins, predictive modelling was conducted using I-

TASSER (Section 2.10.5) and the outputs analysed in PyMOL for viewing the 3D

structure and predicted functional domains such as the TRD and HD of the PINA and

PINB. Modelling of wild type PINA (Figure 4.23) showed very similar structures to

that predicted by Lesage et al. (2011) (Figure 1.5). The model showed homology to the

2S albumin storage protein and exhibited four alpha helices, with the TRD located as an

extended loop between α-helices 1 and 2, as reported previously by Marion et al.

(1994). The five tryptophan residues (W) in PINA and three in PINB with their indole-

ring are shown in blue (Figures 4.23 and 4.24).

An earlier prediction by using Kyte-Doolittle Hydropathy plot had shown strong

hydrophobic properties for the sequence at position 75-80 in the mature PINA protein,

this distinct hydrophobic domain (HD) formed a separate loop between α-helices 3 and

4 (Alfred, 2013). These HDs for PINA and PINB are shown in purple (Figures 4.23 and

4.24) and the phenylalanine residues (F) are marked. The C-score is a confidence score

for estimating the quality of predicted models by I-TASSER (Roy et al., 2010; Zhang,

2008) (Section 2.10.5). The calculated C-score was 0.28 for PINA and 0.37 for PINB,

signifying a model with a high confidence.

Sequence

20 40 60 80 100 120

| | | | | |

DVAGGGGAQQCPVETKLNSCRNYLLDRCSTMKDFPVTWRWWKWWKGGCQELLGECCSRLGQMPPQCRCNIIQGSIQGDLGGIFGFQRDRASKVIQEAKNLPPRCNQGPPCNIPGTIGYYW

Prediction CCCCCCCCCCCCHHHHHCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHCCHHHHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHCCCHCCCCCCCCCCCCCCCCCC

Conf.Score 987766545883666650799999996743113653334511253416999999999888388877359999999999987750140079999999865621029998887788777779

Figure 4.23 Structure prediction using PyMOL and prediction secondary structure for PINA. Prediction line, elements are shown as H for Alpha helix and C for Coil) using I-TASSER for PINA. Structure displaying TRD (blue) and HD (purple) domains are enclosed.

W

W W

W

W

F

Sequence

20 40 60 80 100

| | | | |

EVGGGGGSQQCPQERPKLSSCKDYVMERCFTMKDFPVTWPTKWWKGGCEHEVREKCCKQLSQIAPQCRCDSIRRVIQGRLGGFLGIWRGEVFKQLQRAQSLPSKCNMGADCKFPSGYYW

Prediction CCCCCCCCCCCCHHCHCCCHHHHHHHHHCCCCCCCCCCHHHHHHHCCCHHHHHHHHHHHHHHCCHHHCHHHHHHHHHHHHHHHCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCC

Conf.Score 98776743248053010038999999977433357510347874152179999999999985766346299999999998735054303899999998647510299999988888779

Figure 4.24 Structure prediction using PyMOL and prediction secondary structure for PINB. Prediction line, elements are shown as H for Alpha helix and C for Coil) using I-TASSER for PINB. Structure displaying TRD (blue) and HD (purple) domains are enclosed.

W

W W

F


- 152 -

The COTH (Threading-recombination approach) software (Section 2.10.6) was also

used to predict the possible structure of PINA and PINB protein-protein complex. The

quality of the complex template was detected by TM-score, alignment coverage, and

complex root mean-square deviation (Mukherjee and Zhang, 2011). TM-score was

used for quality assessment of PIN structure predictions, since it has ability in

combining alignment accuracy and coverage. First rank of the template for PIN

proteins was selected when TM-score was determined as individual protein; templates

>0.5 but the complex TM-score was <0.4. The prediction complex model for wild type

PINA and PINB with coverage of alignment, 0.816 is given in Figure 4.25. These

models were determined also for PINA∆HD and PINB∆HD with coverage of

alignment, 0.795 (Figure 4.26). Interestingly, coverage of the alignment which is based

on dividing the length of the alignment by the total length of the query complex is

higher for interaction of wild type PINs than for mutant proteins. The top models

predicted by COTH for interaction of PINA∆HD+PINB∆HD showed change in protein

folding that can lead to aggregation. PIN proteins without HD were folded somehow

differently and the TRD locations in both PIN proteins were changed (Figure 4.26).

The complex structure when the HD was removed exhibited a change in position of the

two tryptophan residues (W40 and W41) (shown in blue) in PINA and proline and

valine residues (P36 and V37) (shown in blue) in PINB. The change in position of the

TRD in predicted PINA∆HD and PINB∆HD complexes may explain the results of lack

of protein-protein interactions in plant cells when the HD is removed. The result shows

that both the TRD and HD regions may have roles in the interactions of PIN proteins,

but may contribute in different ways to the stability of the protein complexes.

DVAGGGGAQQCPVETKLNSCRNYLLDRCSTMKDFPVTWRWWKWWKGGCQELLGECCSRLGQMPPQCRCNIIQGSIQGDLGGIFGFQRDRASKVIQEAKNLPPRCNQGPPCNIPGTIGYYWEVGGGGGSQQCPQERPKLSSCKDYVMERCFTMKDFPVTWPTKWWKGGCEHEVREKCCKQLSQIAPQCRCDSIRRVIQGRLGGFLGIWRGEVFKQLQRAQSLPSKCNMGADCKFPSGYYW Figure 4.25 Modeling result of COTH for PINA and PINB Complex in horizontal and vertical direction. Wild type PINA (green) and PINB (red) complex; two residues of TRD domain for both proteins are positioned in blue; HD domains for both PINA and PINB proteins are bordered of protein sequences.

W

W

P

V

DVAGGGGAQQCPVETKLNSCRNYLLDRCSTMKDFPVTWRWWKWWKGGCQELLGECCSRLGQMPPQCRCNIIQGSQRDRASKVIQEAKNLPPRCNQGPPCNIPGTIGYYWEVGGGGGSQQCPQERPKLSSCKDYVMERCFTMKDFPVTWPTKWWKGGCEHEVREKCCKQLSQIAPQCRCDSIRRLGIWRGEVFKQLQRAQSLPSKCNMGADCKFPSGYYW Figure 4.26 Modeling result of COTH for PINA∆HD and PINB∆HD Complex. HD deletion mutation PINA∆HD (green) and PINB∆HD (red) complex; two residues of TRD domain are positioned in blue

W W

P

V


- 155 -

4.3 Discussion

Previously PIN proteins have been expressed successfully in maize with the 27 kDa γ-

Zein promoter and the 35S polyadenylation region terminator (Zhang et al., 2009).

RNA-mediated silencing (RNAi) of the Pin genes in transgenic wheat reported with

stable transformation via A. tumefaciens under control of 35S promoter (Gasparis et al.,

2011). Moreover recently overexpression of Pina gene in transgenic durum wheat was

conducted under the control of the maize Ubiquitin promoter and also under the control

of 35S promoter (Li et al., 2014). In the present work, the PIN proteins for BiFC assay

have been expressed successfully under the control of 35S promoter.

Protein-protein interaction is one of the most important considerations for the function

of many proteins. The present work is the first investigation of in vivo interaction for

PIN proteins based on the BiFC assay in a plant system. Defining protein interactions

can be assisted by BiFC assays in live plant cells such as N. benthamiana (Goodin et al.,

2008; Ohad et al., 2007; Waadt et al., 2008). This approach is based on the

complementation between two non-fluorescent fragments of the yellow fluorescent

protein (YFP) when they are brought together by interactions between proteins fused to

each fragment resulting in fluorescence (Hu and Kerppola, 2003; Kerppola, 2006).

Since the induction of the fluorescence signal is dependent on the physical association

between the two terminal parts of YFP, it is dependent not only on their presence but

also their orientation. Overall, when the structural basis of a protein complex is not

clear, as in the present case, the two interacting proteins should be fused N- and also C-

terminally to the C- and N- terminal parts of YFP and all eight combinations tested for

fluorescence complementation (Kerppola, 2006). Table 4.1, shows interactions results

in different directions for heterodimersations and homodimersations, for both PINA and

PINB. Strong interactions were observed only in one direction: PINA-YC+YN-PINB,

i.e., co-expression of the C-terminal half of YFP fused to PINA and the N-terminal half

of YFP fused to PINB. The finding suggests that the orientation of the PIN proteins is

important for their interactions. Previous work by Ziemann et al. (2008) using the yeast

two-hybrid system (Fields and Song, 1989) showed physical interactions between wild

type of PINA and PINB, and also PINB interacting with itself while PINA showed

weaker self interaction. Further, the important role of direction for PIN proteins in the


- 156 -

yeast two-hybrid system confirmed in studies of wild type as well as the mutated (TRD

and HD domain-deleted) proteins (Alfred, 2013; Alfred et al., 2014).

Using BiFC, leaf cells of N. benthamiana showed fluorescence signal due to PINB

homo-dimerisation (YN-PINB+YC-PINB). The presence of three tryptophan residues

in the TRD of PINB may explain the result. However, most interestingly, no

interactions were detected for PINA homo-dimerisation, suggesting the domain alone or

its tryptophans (five in PINA) may not be directly relevant to the interactions.

Fluorescence complementation can occur when the YFP fragments are fused to

positions that are separated by an average distance of greater than 100A°, which

provides enough flexibility to allow reconstitution of the split-YFP fragments (Hu et al.,

2002). The lack of fluorescence of PINA homo-dimerisation may result from

insufficient flexibility compared to that of PINB complexes.

The region or amino acid residues involved in the PIN proteins interactions still remains

elusive. The TRD is the most important region for lipid interactions (Kooijman et al.,

1997; Kooijman et al., 1998; Jing et al., 2003), therefore one strong candidate is the

TRD in both proteins, at residues 34-46 in mature PINA and 35-47 in mature PINB.

The other candidate is the little-studied hydrophobic domain (HD) at position of residue

75-85 of the mature PINA (Le Bihan et al., 1996), and the corresponding residues 75-83

of the mature PINB. Clones created previously (Alfred, 2013; Alfred et al., 2014) by

site-directed mutagenesis (SDM) PCR to delete the TRD within PINA and the HD

within PINA and PINB were therefore used in the present work. The PINA∆HD

interactions with PINB∆HD showed no fluorescence signal based on BiFC assays, in

contrast to interaction of a specific orientation of the wild type PINA and PINB. The

interactions for PINA and PINB without HD tested in the same orientation were

unsuccessful in inducing fluorescence. Furthermore, no fluorescence signal was

observed with itself or between PINB∆HD and wild type PINB. The results indicate

that the HD domains are important for co-expression and/or stability and/or interaction

between PINA and PINB and among PINB and PINB in N. benthamiana leaf cells.

There is one possible explanation for the non-interaction of PINA∆HD+PINB∆HD;

aggregation of mutant proteins, due to the change in the protein folding.


- 157 -

Deletion of the HD may also have an effect on the refolding of the PINs. Dimerisation

and/or oligomerisations can have different structural and functional advantages for

proteins, such as improved stability, control over accessibility and specificity of active

sites, and increased complexity (Marianayagam et al., 2004). Protein complexes are

stabilized by the burial of hydrophobic complementary occluding surfaces, covalent

bonds such as disulphide bridges, and hydrogen bonding (Lesk, 2010). The disulphide

bonds are indeed necessary to stabilize the α-helical structure and maintain the native

structure and solubility of PINA and PINB (Le Bihan et al, 1996). The formation of α-

helices at membrane surfaces is also an important point for binding to membrane

(Seelig, 2004). The preliminary protein-protein complex predictions based on the

COTH have shown refolding and a shift in the rotation of structure of the mutant PINs,

which may affect their functionality.

No protein-protein interactions were noted for PINA∆TRD with wild type PINB. Thus

the TRD of PINA may be important for expression in planta and protein-protein

interactions. Previous studies have emphasized that the TRD region has important role

in binding to the membranes polar lipids on the surface of starch (Blochet et al., 1993;

Kooijman et al., 1997; Giroux and Morris, 1997), direct binding to surface of starch

granules (Wall et al., 2010) and the activity of PINs (Capparelli et al., 2005; Dubreil et

al., 1998; Jing et al., 2003). The TRD has also been shown to be important for the

antibacterial and antifungal activities of PINs (Jing et al., 2003; Miao et al., 2012;

Phillips et al., 2011). The self-assembly of PINA micelle in solution has been shown by

hydrophobic forces that involve intermolecular interactions between the tryptophan

residues in the TRD (Clifton et al., 2011a). However no such self-assembly has been

observed for PINB, possibly due to fewer hydrophobic tryptophan residues in its TRD

(Clifton et al., 2011a; Pauly et al., 2013). There is evidence that PINA forms

aggregates, and TRD has been implicated in the formation of PINA aggregates (Le

Bihan et al., 1996). Thus, PINA-PINB interaction may explain the reported change to

hard grain when there is a mutation or a lack in either PIN protein (Giroux and Morris,

1997, 1998). Transgenic wheat work has provided further insights into the roles of

PINA and PINB together and individually in influencing grain texture (Giroux and

Morris, 1998; Morris et al., 2001; Wanjugi et al., 2007) (reviewed in Section 1.5.4).


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Two tryptophan residues (W41 and W44) at the TRD have shown essential role for

interaction of PINA with the yeast plasma membranes, while the lysine in the TRD of

PINB seems to be more important (Evrard et al., 2008). In planta mutagenesis studies

had found that mutations within the TRD of PINA and PINB resulted significantly

increased the grain hardness, and PINB function was likely to be more critical to overall

Ha locus effects (Feiz et al., 2009a). The TRD has a role in stabilizing the structure of

both PIN proteins (Pauly et al., 2013).

The present work has shown that the PINA and PINB proteins do interact physically in

planta, and PINB can also self-associate, indicating that its function may more

considerable despite having a TRD with fewer tryptophan residues. A more detailed

understanding of the mechanisms for protein-protein interaction between the PIN

proteins is required to explain many of their co-operative and inter-dependant properties

involved in starch binding and weather these are also relevant to their bioactivity against

microbial pathogen.

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CHAPTER 5

General discussion and future directions

Chapter 5 General discussion and future directions

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5.1 General discussion

Wheat is the third most widely consumed cereals in the world after maize and rice. The

quality of wheat is critical in the food industry owing to its nutritional value and health

benefits. The hardness or texture of the wheat grain is a major determinant of the

quality, as it affects milling and end use properties.

The first chapter of this thesis presented a comprehensive review of literature on

structure of the wheat grain, grain hardness and its effects on the end-use of wheat and

the discoveries of friabilin and puroindoline proteins. Friabilin is a 15 kDa protein

complex that consists of two structurally similar proteins, puroindoline-a (PINA) and

puroindoline-b (PINB). It was originally suggested that friabilin act as a non-stick

surface between the starch granules and the protein matrix to determine wheat grain

hardness. The PINs are unique small proteins with a highly conserved 10 cysteine-

backbone and a tryptophan-rich domain (TRD), the latter most likely involved in the

binding to the phospholipids on the surface of starch granules. The soft grain texture in

wheat encoded by the wild type Pin alleles (Pina-D1a and Pinb-D1a), while the hard

texture occurs as a result of mutation in one or both of the Pin genes, and deletions of

both genes result in the ‘very hard’ texture of durum wheat. Chapter 1 also described

evidence of transgenic Pin genes altering grain texture, as well as plant-based

expression systems for production of valuable recombinant proteins such as anti-

microbial peptides (AMPs). The review also presented several predicted homology

models of PIN proteins due to the lack of an absolute three dimensional structure.

Another limitation in structural analysis of PINs is their strong aggregation properties at

high protein concentration. Several other gaps in literature were also identified, such as

the in vivo roles of PIN proteins within the wheat seed and the exact mode of actions of

PINs in grain hardness and their co-operative activity at membranes. Hence,

characterisation of PIN proteins is critical to exploit the potential benefits of PIN

proteins and PIN-based peptides in agricultural or medical applications. Therefore, the

major objectives of this research were as follows:


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1. To clone the Pin genes encoding the putative mature PINA and PINB in magnICON®

viral vector system and A. tumefaciens infiltrations, to express and optimize their

transient expression for high yield in the leaves of Nicotiana benthamiana.

2. To test the antibacterial and antifungal activities of recombinant PIN proteins purified

with affinity purification using His-tag and hydrophobic interactions systems.

3. To analyse the protein-protein interactions among the PIN proteins in planta using

the BiFC system based on pNBV vectors and co-expression of the silencing suppressor

protein p19.

4. To test the significance of the tryptophan-rich domain (TRD) of PINA and the

hydrophobic domain (HD) of both PINs in any interactions between the PINs using the

BiFC system.

The above aims were addressed using a number of experimental approaches as set out in

Chapter 2. In brief, these included molecular and biochemical methodologies such as,

DNA extractions, PCRs, cloning into viral vectors, plasmid purifications, DNA

sequencing, electroporation of A. tumefaciens cells, agroinfiltration, in planta protein

expression, protein purification and quantitation, SDS-PAGE, western blot and ELISA

analyses, fluorescence microscopy, microbiological techniques and also bioinformatics

tools (e.g., DNA and protein sequence alignments, primer design, BLAST searches, 3D

modeling and protein-protein complex structure predications).

The aims 1 and 2 were addressed in Chapter 3. In brief, this chapter investigated the

plant expression system as a platform to produce recombinant PINA and PINB proteins

in sufficient yield for antimicrobial activities. The study focussed on the rapid transient

expression of PINs using the deconstructed tobacco mosaic virus-based ‘magnICON®’

for subcellular localisation of PINs in different compartments of N. benthamiana. The

results have shown: (i) the presence of recombinant PINA and PINB in both monomeric

and oligomeric forms when targeted to the chloroplast, cytosol and ER as shown by

western blot and ELISA analyses (ii) PIN A and PINB yields of up to 0.8% TSP or 51.7


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µg/g of fresh weight for PINA, and 70 µg/g of fresh weight for PINB when proteins

targeted to the chloroplast (iii) the achievement of maximum yields of recombinant PIN

proteins at 10 dpi (iv) heat-sensitivity of recombinant PIN proteins (v) establishment of

the most efficient purification method for recombinant PINs based on hydrophobic

interaction (vi) antimicrobial activity for recombinant PINA and PINB against

Escherichia coli (a Gram negative bacterium) and Staphylococcus aureus (a Gram

positive bacterium) and four phytopathogenic fungal strains and low haemolytic activity

towards mammalian cells. Overall, the work has led to successful expression,

purification and characterisation methodology for obtaining PIN proteins with

appropriate quality that is essential for potential infection control and therapeutic

applications.

Aims 3 and 4 were addressed in Chapter 4. There is evidence that PINs may interact

co-operatively or interdependently to confer the soft grain texture. The present work

showed interactions between PINA and PINB proteins using BiFC system in N.

benthamiana leaves, just in one direction; PINA-YC+YN-PINB, based on the

fluorescence intensity visualised in living plant cells. The finding suggests that the

orientation of the PIN proteins is important for their interactions. In addition,

homodimerisation was noted for PINB (YN-PINB+YC-PINB) but not PINA. Moreover

the chapter investigated the key regions/residues essential for protein-protein

interaction. Site-directed mutagenesis (SDM) PCR had been conducted previously to

deletions in PINA at the TRD and in both PINs at the hydrophobic domain (HD). These

inserts were subcloned into the BiFC vectors in the present work. The results indicate

that no interaction occurred between PINA and PINB, or between PINB and PINB,

without their HD. The HD domain may affect the folding of PIN proteins and rotation

possibilities of the structure. Predictions of the 3D models of the possible structure for

PINs further corroborated this hypothesis. The deletion of the TRD in PINA also

showed no interaction with wild type PINB. Overall, the result shows that both the

TRD and HD regions may have roles in the in planta interactions of PIN proteins, but

the two domains may contribute in different ways to stability of the protein complexes.

The mutant PIN proteins were also expressed in planta based on magnICON® system to

test their expression. The result showed that PINs without the HD were successfully


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expressed, while the PINA without TRD was not detected. The results suggest that

PINA with deletion of TRD may be degraded, or the Durotest antibody was not able to

detect this mutant protein, suggesting the TRD is among the epitopes for this antibody.

5.2 Future directions

The significance of the PIN proteins in the wheat industry has led to a considerable

amount of research focus on their structures and mechanisms. The worldwide research

has led to over 150 publications that outline their lipid-binding properties, the mutant

alleles as well as orthlogs in other monocot crop species, also the antimicrobial

properties of the proteins and the peptides based on these. However, the exact

mechanisms of action for PINs are not completely clear yet. The results of the present

study led to successful expression of bioactive proteins in a plant systems and also

establishe PINA-PINB and PINB-PINB associations in planta, as summarized above.

However further in-depth research is required on nature and mechanisms for their

functionality. The identity of the active protein can be established using peptide

mapping of the trypsinized protein, sizing using MALDI-TOF MS methods and identity

through LC–MS/MS (Klimyuk et al., 2014). Additionally, it would be important to use

circular dichroism (CD) tools (Greenfield, 2007) to determine the secondary structure

and folding properties of recombinant proteins to assess how the different mutations

affects their structure and stability.

Designing more strategies for increased production of soluble PIN proteins, such as

plant-codon optimisation would be important, as effected on increasing yield of protein

in plant system (Webster et al., 2009). The choice of expression systems such as cell-

free system (Endo and Sawasaki, 2006; Sawasaki et al., 2002), or vectors with fusion

tag, for instance, Halo-tag (Los et al., 2008) would be important. Furthermore, several

fusion strategies have been developed recently to significantly enhance the production

yield of plant-made recombinant proteins, as reviewed by Conley et al. (2011).

The exact mechanism of membrane activity for PIN-based peptides and proteins still is

unclear. Hence it would be useful to design chloroplast transformation vector with the

PIN-based peptide fused with GFP to confer stability to the peptide (Lee et al., 2011b).


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PIN-based peptides can be obtained with the tag to help their purification for further

analysis of direct interactions with biological membranes. Expression and

characterisation of antimicrobial peptides in chloroplasts to control viral and bacterial

infections has been successfully utilised (Daniell et al., 2009a; Lee et al., 2011b; Nadai

et al., 2009; Ruhlman et al., 2007; Sanz-Barrio et al., 2011). Further work is needed to

explore these applications for PINs, especially for activity against pathogens of

relevance to the food and agriculture industries.

This study has shown protein-protein interaction of PIN proteins in planta. The

knowledge gained will be invaluable for future work for more detailed understanding of

the mechanisms interactions, which is required to explain their co-operative or

interdependent properties in starch binding. Site-directed mutagenesis of residues that

may have a role in the hardness phenotype would be important for exploring their

effects on protein interaction. Further, the GSP-1 and PINB-2v1 proteins may be

additional factors that interact with the PINs, contribute to further variation in wheat

grain hardness, and therefore would be important for exploring their effects on protein

interaction. Flow cytometry techniques (Berendzen et al., 2012; Morell et al., 2008) and

multicolour BiFC analysis may allows simultaneous visualisation of multiple protein

complexes in the same cell (Hu and Kerppola, 2003; Kerppola, 2008) and enable

subcellular localisation of the interaction (Kerppola, 2013). Furthermore, quantification

of BiFC fluorescence can be measured by analytical flow cytometry (Berendzen et al.,

2012).

The PIN proteins were discovered more than twenty years ago and numerous studies so

far have already revealed a number of their fundamental biochemical and functional

aspects. Further investigations building on this wealth of knowledge will one day

explain the unique activities and effects of these small proteins.

References

- 165 -

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Appendices

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Appendices

Appendix I

Table I.1 Primer used for deletion of HD in PINA and PINB; TRD in PINA

* F: Forward primer; R: Reverse primer

Primer* Sequence (5’ to 3’) Application PINA∆TRD-F

PINA∆TRD-R

5’-atgaaggatGGTTGTCAAGAGCTCCTTGG-3’ Deletion of TRD in PINA (position 34-46) 5’-ttgacaaccATCCTTCATCGTTGAGCATC-3’

PINA∆HD-F

PINA∆HD-R

5’-caggggtcaCAGCGTGATCGGGCAAGCAA-3’ Deletion of hydrophobic domain (HD) in PINA (position 75-85) 5’-atcacgctgTGACCCCTGGATGATGTTGC-3’

PINB∆HD-F PINB∆HD-R

5’-atccggcgaTTGGGCATTTGGCGAGGTGA-3’ Deletion of hydrophobic domain (HD) in PINB (position 75-83) 5’-aatgcccaaTCGCCGGATAGAATCACAGC-3’

Appendices

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Appendix II

Figure II.1 pICH-PINA expressions constructs. DNA sequence of mature putative Pina-D1a (DQ363911, line1) aligned with the sequence of pICH-PINA-His clone (line 2) and with the sequence of pICH-PINA-His-SEKDEL clone (line 3). His-tag and SEKDEL site underlined.

Figure II.2 pICH-PINA expressions construct. Amino acid sequence of mature putative PINA (line1) aligned with the sequence of pICH-PINA-His clone (line 2) with sequence of pICH-PINA-His-SEKDEL clone (line 3).

10 20 30 40 50 60. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature ~ ~ ~ ~ ~ ~ ~ ~ ~ G A T G T T G C T G G C G G G G G T G G T G C T C A A C A A T G C C C T G T A G A G A C A A A G C T ApICH-PINA-His A A T T C G A T G G A T G T T G C T G G C G G G G G T G G T G C T C A A C A A T G C C C T G T A G A G A C A A A G C T ApICH-PINA-His-SEKDEL A A T T C G A T G G A T G T T G C T G G C G G G G G T G G T G C T C A A C A A T G C C C T G T A G A G A C A A A G C T A

70 80 90 100 110 120. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature A A T T C A T G C A G G A A T T A C C T G C T A G A T C G A T G C T C A A C G A T G A A G G A T T T C C C G G T C A C CpICH-PINA-His A A T T C A T G C A G G A A T T A C C T G C T A G A T C G A T G C T C A A C G A T G A A G G A T T T C C C G G T C A C CpICH-PINA-His-SEKDEL A A T T C A T G C A G G A A T T A C C T G C T A G A T C G A T G C T C A A C G A T G A A G G A T T T C C C G G T C A C C

130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature T G G C G T T G G T G G A A A T G G T G G A A G G G A G G T T G T C A A G A G C T C C T T G G G G A G T G T T G C A G TpICH-PINA-His T G G C G T T G G T G G A A A T G G T G G A A G G G A G G T T G T C A A G A G C T C C T T G G G G A G T G T T G C A G TpICH-PINA-His-SEKDEL T G G C G T T G G T G G A A A T G G T G G A A G G G A G G T T G T C A A G A G C T C C T T G G G G A G T G T T G C A G T

190 200 210 220 230 240. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature C G G C T C G G C C A A A T G C C A C C G C A A T G C C G C T G C A A C A T C A T C C A G G G G T C A A T C C A A G G CpICH-PINA-His C G G C T C G G C C A A A T G C C A C C G C A A T G C C G C T G C A A C A T C A T C C A G G G G T C A A T C C A A G G CpICH-PINA-His-SEKDEL C G G C T C G G C C A A A T G C C A C C G C A A T G C C G C T G C A A C A T C A T C C A G G G G T C A A T C C A A G G C

250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature G A T C T C G G T G G C A T C T T C G G A T T T C A G C G T G A T C G G G C A A G C A A A G T G A T A C A A G A A G C CpICH-PINA-His G A T C T C G G T G G C A T C T T C G G A T T T C A G C G T G A T C G G G C A A G C A A A G T G A T A C A A G A A G C CpICH-PINA-His-SEKDEL G A T C T C G G T G G C A T C T T C G G A T T T C A G C G T G A T C G G G C A A G C A A A G T G A T A C A A G A A G C C

310 320 330 340 350 360. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature A A G A A C C T G C C G C C C A G G T G C A A C C A G G G C C C T C C C T G C A A C A T C C C C G G C A C T A T T G G CpICH-PINA-His A A G A A C C T G C C G C C C A G G T G C A A C C A G G G C C C T C C C T G C A A C A T C C C C G G C A C T A T T G G CpICH-PINA-His-SEKDEL A A G A A C C T G C C G C C C A G G T G C A A C C A G G G C C C T C C C T G C A A C A T C C C C G G C A C T A T T G G C

370 380 390 400 410. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .

Pina-Mature T A T T A C T G G T G A pICH-PINA-His T A T T A C T G G C A C C A T C A C C A T C A C C A T T G A G G A T C C pICH-PINA-His-SEKDEL T A T T A C T G G C A C C A T C A C C A T C A C C A T T C T G A G A A A G A T G A G C T A T G A G G A

10 20 30 40 50 60. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature ~ ~ ~ D V A G G G G A Q Q C P V E T K L N S C R N Y L L D R C S T M K D F P V T W R WW K WW K G G C Q E L L G E C C SpICH-PINA-His N S M D V A G G G G A Q Q C P V E T K L N S C R N Y L L D R C S T M K D F P V T W R WW K WW K G G C Q E L L G E C C SpICH-PINA-His-SEKDEL N S M D V A G G G G A Q Q C P V E T K L N S C R N Y L L D R C S T M K D F P V T W R WW K WW K G G C Q E L L G E C C S

70 80 90 100 110 120. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pina-Mature R L G Q M P P Q C R C N I I Q G S I Q G D L G G I F G F Q R D R A S K V I Q E A K N L P P R C N Q G P P C N I P G T I GpICH-PINA-His R L G Q M P P Q C R C N I I Q G S I Q G D L G G I F G F Q R D R A S K V I Q E A K N L P P R C N Q G P P C N I P G T I GpICH-PINA-His-SEKDEL R L G Q M P P Q C R C N I I Q G S I Q G D L G G I F G F Q R D R A S K V I Q E A K N L P P R C N Q G P P C N I P G T I G

130. . . . | . . . . | . . . . | . .

Pina-Mature Y YW * pICH-PINA-His Y YW H H H H H H * G S pICH-PINA-His-SEKDEL Y YW H H H H H H S E K D E L * G

Appendices

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Figure II.3 pICH-PINB expressions construct. DNA sequence of mature putative PINB aligned with the sequence of pICH-PINB-His clone (line 2) and with sequence of pICH-PINB-His-SEKDEL clone (line 3). His-tag and SEKDEL site underlined.

Figure II.4 pICH-PINB-His expressions construct. Amino acid sequence of mature putative PINB (line1) aligned with the sequence of pICH-PINB-His clone (line 2) with sequence of pICH-PINB-His-SEKDEL clone (line 3).

10 20 30 40 50 60. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature ~ ~ ~ ~ ~ ~ G A A G T T G G C G G A G G A G G T G G T T C T C A A C A A T G T C C G C A G G A G C G G C C G A A G C T ApICH-PINB-His A C C A T G G A A G T T G G C G G A G G A G G T G G T T C T C A A C A A T G T C C G C A G G A G C G G C C G A A G C T ApICH-PINB-His-SEKDEL T C G A T G G A A G T T G G C G G A G G A G G T G G T T C T C A A C A A T G T C C G C A G G A G C G G C C G A A G C T A

70 80 90 100 110 120. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature A G C T C T T G C A A G G A T T A C G T G A T G G A G C G A T G T T T C A C A A T G A A G G A T T T T C C A G T C A C CpICH-PINB-His A G C T C T T G C A A G G A T T A C G T G A T G G A G C G A T G T T T C A C A A T G A A G G A T T T T C C A G T C A C CpICH-PINB-His-SEKDEL A G C T C T T G C A A G G A T T A C G T G A T G G A G C G A T G T T T C A C A A T G A A G G A T T T T C C A G T C A C C

130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature T G G C C C A C A A A A T G G T G G A A G G G C G G C T G T G A G C A T G A G G T T C G G G A G A A G T G C T G C A A GpICH-PINB-His T G G C C C A C A A A A T G G T G G A A G G G C G G C T G T G A G C A T G A G G T T C G G G A G A A G T G C T G C A A GpICH-PINB-His-SEKDEL T G G C C C A C A A A A T G G T G G A A G G G C G G C T G T G A G C A T G A G G T T C G G G A G A A G T G C T G C A A G

190 200 210 220 230 240. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature C A G C T G A G C C A G A T A G C A C C A C A A T G T C G C T G T G A T T C T A T C C G G C G A G T G A T C C A A G G CpICH-PINB-His C A G C T G A G C C A G A T A G C A C C A C A A T G T C G C T G T G A T T C T A T C C G G C G A G T G A T C C A A G G CpICH-PINB-His-SEKDEL C A G C T G A G C C A G A T A G C A C C A C A A T G T C G C T G T G A T T C T A T C C G G C G A G T G A T C C A A G G C

250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature A G G C T C G G T G G C T T C T T G G G C A T T T G G C G A G G T G A G G T A T T C A A A C A A C T T C A G A G G G C CpICH-PINB-His A G G C T C G G T G G C T T C T T G G G C A T T T G G C G A G G T G A G G T A T T C A A A C A A C T T C A G A G G G C CpICH-PINB-His-SEKDEL A G G C T C G G T G G C T T C T T G G G C A T T T G G C G A G G T G A G G T A T T C A A A C A A C T T C A G A G G G C C

310 320 330 340 350 360. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature C A G A G C C T C C C C T C A A A G T G C A A C A T G G G C G C C G A C T G C A A G T T C C C T A G T G G C T A T T A CpICH-PINB-His C A G A G C C T C C C C T C A A A G T G C A A C A T G G G C G C C G A C T G C A A G T T C C C T A G T G G C T A T T A CpICH-PINB-His-SEKDEL C A G A G C C T C C C C T C A A A G T G C A A C A T G G G C G C C G A C T G C A A G T T C C C T A G T G G C T A T T A C

370 380 390 400. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . .

PINB-Mature T G G T G A pICH-PINB-His T G G C A C C A T C A C C A T C A C C A T T G A C T C G A G G C pICH-PINB-His-SEKDEL T G G C A C C A T C A C C A T C A C C A T T C T G A G A A A G A T G A G C T A T G A G G A T C C

10 20 30 40 50 60. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature ~ ~ E V G G G G G S Q Q C P Q E R P K L S S C K D Y V M E R C F T M K D F P V T W P T K WW K G G C E H E V R E K C C KpICH-PINB-His T M E V G G G G G S Q Q C P Q E R P K L S S C K D Y V M E R C F T M K D F P V T W P T K WW K G G C E H E V R E K C C KpICH-PINB-His-SEKDEL S M E V G G G G G S Q Q C P Q E R P K L S S C K D Y V M E R C F T M K D F P V T W P T K WW K G G C E H E V R E K C C K

70 80 90 100 110 120. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

PINB-Mature Q L S Q I A P Q C R C D S I R R V I Q G R L G G F L G I W R G E V F K Q L Q R A Q S L P S K C N M G A D C K F P S G Y YpICH-PINB-His Q L S Q I A P Q C R C D S I R R V I Q G R L G G F L G I W R G E V F K Q L Q R A Q S L P S K C N M G A D C K F P S G Y YpICH-PINB-His-SEKDEL Q L S Q I A P Q C R C D S I R R V I Q G R L G G F L G I W R G E V F K Q L Q R A Q S L P S K C N M G A D C K F P S G Y Y

130. . . . | . . . . | . . . . | .

PINB-Mature W * pICH-PINB-His W H H H H H H * L E pICH-PINB-His-SEKDEL W H H H H H H S E K D E L * G S

Appendices

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Appendix III Protein identification using MALDI-TOF

To obtain an accurate mass for recombinant PINs, and further investigate multimer

formation, MALDI-TOF analysis was done. The result was inconclusive as data

provide unknown peptides sequence.

The protocol for MALDI-TOF was based on Shevchenko et al. (2007) with minor

modification and was applied to SDS-PAGE bands of interest at various stages of

purification. Coommassie stained gels were used for protein identification. Protein

bands of PINs resolved on SDS-PAGE were excised from the gel using a sterile scalpel

and transferred into microcentrifuge tubes. The gel plugs were first added in 500 µL of

neat acetonitrile by incubating at 37°C for 10 minutes until the gel pieces shrank. The

gel plug solution was removed after centrifugation at 10,000×g, then 100 µL DTT was

added to cover the gel pieces and incubated for 30 minutes at 56°C. The tubes were

allowed to cool down to room temperature for 10 minutes and solution was removed.

50 µL of iodoacetamide solution added to tubes and incubated for 20 minutes at RT in

dark. Then 500 µL of acetonitrile were added and incubated for 10 minutes. Then 100

µL of solution (50% v/v acetonitrile and 50% v/v 100 mM ammonium bicarbonate) was

added and tubes incubated at RT with occasional vortex for 2 hours. 50 µL of the

trypsin solution (Biolabs, Australia) was added to each sample then added 20 µL

ammonium bicarbonate buffer (100mM) and incubate at 37°C for overnight. The next

day tubes were incubated at room temperature and the gel pieces spun down. Then 100

µL extraction buffer (5% formic acid: acetonitrile 1:1 (v/v)) was added to each tube and

incubated for 15 minutes at 37°C on a shaker. The supernatant was collected into new

tube and dried down in vacuum centrifuge for 2 hours at 50°C. Samples were

resuspended in trifluoroacetic acid and then with equal volume of saturated α-Cyano-4-

hydroxycinnamic acid (CHCA) matrix was spotted onto target plate of Axima MALDI-

TOF mass spectrometer (Shimadzu-Biotech, Japan). Samples were analysed using the

Shimadzu MALDI-MS software. A total of four sample spots (1 μL each) were

analysed for each aliquot taken.

Appendices

- 204 -

Appendix IV

Figure III.1 Partial sequence of pNBV constructs. A: pNBV-gene-YC; B: pNBV-gene-YN 35S promoter highlighted in gray. The two NotI (GCGGCCGC) site highlighted in red; XbaI site [TCTAGA] and XmaI [CCCGGG] highlighted; DNA sequence of YC and YN highlighted in blue. Sequence of NosT and LacZ highlighted.

A

B

Appendices

- 205 -

Figure III.2 Partial Sequence of pNBV constructs. A: pNBV-YC-gene; B: pNBV-YN-gene 35S promoter highlighted in gary. The two NotI (GCGGCCGC) site highlighted in red; EcoRI site [GAATTC] and BamHI [GGATCC] highlighted; DNA sequence of YC and YN highlighted in blue. Sequence of NosT and LacZ highlighted.

A

B

Appendices

- 206 -

Appendix V Record of Proposed Notifiable Low Risk Dealing(s) (NLRDs):

(Regulation 13A (1) (a) - Gene Technology Regulations 2001 (as amended))

INSTRUCTIONS: Under r.13A(1)(c) of the Gene Technology Regulations 2001 (as amended), the Institutional Biosafety Committee (IBC) must provide a copy of this record to (i) the person or accredited organisation that requested the IBC to assess the proposed dealing; and (ii) the project supervisor for the proposed dealing. Under r.13A (2)(a)(i) an accredited organisation that proposed the dealing must include a copy of the IBC's record with the Accredited Organisation Annual Report to the Gene Technology Regulator. Under r.13A (2)(a)(ii) non-accredited organisations must provide a copy of this record to the Regulator at the end of the financial year.

NOTE: This form also captures information required by the Gene Technology Regulator to meet the requirements of r. 39 for NLRDs in maintaining a record of GMO and GM product dealings.

* References to NLRD types are the paragraph numbers relating to the kinds of dealing detailed in Parts 1 and 2 in Schedule 3 of the Gene Technology Regulations 2001 (as amended). These Regulations are available on the OGTR website <www.ogtr.gov.au>.

IBC NLRD Identifier (number) PC2-N26/10

Date of IBC Assessment 18 November 2010

Name of IBC Monash University IBC #309

Name of Organisation Conducting Dealing

Monash University

Project Title Production, characterisation and evaluation of recombinant puroindolines in plants

PC1 NLRD TYPE (a) – (c)*

PC2 NLRD TYPE (a) – (i)* (b) (ba) (c) (d)

Name(s) of Project Supervisor(s) Dr Diane Webster, Prof Mrinal Bhave

Has the IBC assessed the proposed dealing to be an NLRD?

Yes

Appendices

- 207 -

Publications arising from this work

Poster presentations:

Azadeh Niknejad, Diane Webster, Mrinal Bhave. (10th-14th February 2013);

Recombinant plant-made puroindolines exhibit anti-microbial activity and interaction

in plant system. 38th Lorne conference on protein structure and function, Lorne,

Victoria, Australia.

Azadeh Niknejad, Diane Webster, Mrinal Bhave. (11th July 2013); Investigating PIN

proteins interactions in a plant system using Bimolecular Fluorescence

Complementation (BiFC). 12th Melbourne protein group student symposium, La Trobe

University, Melbourne, Victoria, Australia.

Manuscripts:

Azadeh Niknejad, Diane Webster, Mrinal Bhave. Transient expression and

characterization of puroindoline proteins in Nicotiana benthamiana using a virus-

based expression system. (in preparation)

Azadeh Niknejad, Diane Webster, Mrinal Bhave. Evidence of physical interactions of

puroindoline proteins in living plant cells using bimolecular fluorescence

complementation (BiFC) analysis. (in preparation)

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